Vector co-expressing vaccine and costimulatory molecules

ABSTRACT

Compositions and methods for co-expressing a secretable vaccine protein (such as gp96-Ig) and T-cell co-stimulatory molecules from a single vector, among others, are provided herein. Materials and methods for using gp96-Ig vaccination and T-cell co-stimulation to treat a clinical condition (e.g., cancer) in a subject also are provided.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication Nos. 62/113,153, filed Feb. 6, 2015, and 62/174,942, filedJun. 12, 2015, the entire contents of all of which are herebyincorporated by reference.

TECHNICAL FIELD

This document relates, inter alia, to materials and methods for usingvaccination and T-cell co-stimulation to treat a clinical condition in asubject, including materials and methods for co-expressing a vaccine(e.g. gp96-Ig) and T-cell co-stimulatory molecules from a single vector.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:HTB-021-SequenceListing.txt; date recorded: Feb. 4, 2016; file size: 73KB).

BACKGROUND

Cancer is a disease that arises from a prolonged period of geneticinstability that extends the lifespan of a normal cell. The triggeringevent that marks the beginning of this period is variable between celltypes, but commonly is the acquisition of a mutation in a tumorsuppressor gene such as p53 or Rb, a mutation in a proto-oncogene suchas KRAS or myc, or infection of a cell with an oncogenic virus such asHPV16 or EBV. Whatever the origin, cells that acquire mutations in genesthat enable them to escape normal growth controls or cell death pathwaysbecome more likely to acquire additional mutations. Once a cell hasacquired “enough” mutations, typically thought to be at least six, it nolonger is responsive to intrinsic or extrinsic signals that wouldrestrain its growth or trigger apoptosis.

Because tumors arise from host cells, the body's immune system isinitially tolerant to those cells. The acquisition of tumorigenicmutations may or may not lead to production of a mutated proteincontaining an epitope that is sufficiently non-self to becomeimmunogenic. If a cell acquires an immunogenic mutation, it can besought out and destroyed by the host immune system, a process known asimmunosurveillance (Smyth et al., Adv Immunol 2006, 90:1-50). Murinestudies have provided support for the immune surveillance hypothesis(Dunn et al., Nat Immunol 2002, 3:991-998; Shankaran et al., Nature2001, 410:1107-1111; and Dunn et al., Annu Rev Immunol 2004,22:329-360), and also suggested that innate in addition to so-calledadaptive immune responses may facilitate rejection of immunogenic tumors(Unni et al., Proc Natl Acad Sci USA 2008, 105:1686-1691; Taieb et al.,Nat Med 2006, 12:214-219; and Raulet and Guerra, Nat Rev Immunol 2009,9:568-580). Innate responses can be evoked through induced expression ofNK activating signals such as NKG2D ligand expression or following DNAdamage incurred as a result of mutagenic or viral processes. Some cellsthat acquire immunogenic mutations also gain the capacity to engagenormal immune regulatory systems that dampen anti-self immune responses(Rabinovich et al., Annu Rev Immunol 2007, 25:267-296). The pathwaysdriving the activation of host regulatory mechanisms are poorlyunderstood. Still other cells may gain a number of oncogenic mutationswithout ever producing an immunogenic peptide that leads to activationof the host immune system. Therefore, tumor cells that produce animmunogenic peptide during their transformation must continuously evadeanti-tumor immune responses in order to survive, whereas tumors thatbecome transformed without activating the immune system may not rely onsuch immune regulatory mechanisms for survival.

SUMMARY

It is possible that combination therapies including combinations orsubcombinations of one or more checkpoint inhibitors, one or morevaccines, and one or more T cell costimulatory molecules may expand thebase of cancer patients that can benefit from immunotherapy. Vaccinesmay contribute to this response by increasing both the frequency oftumor-antigen specific CD8+ T cells and also the number of tumorantigens recognized by those CD8+ T cells. T cell costimulatorymolecules may enhance the response by further increasing the frequencyand/or enhancing the activation of tumor antigen-specific T cells, andalso by increasing the expression of tumor-killing effector molecules byCD8+ T cells. When used in combination with checkpoint inhibitors, itmay be possible to generate a broad range of highly activated CD8+ Tcells that will be able to infiltrate tumors and will not be inhibitedby various checkpoint pathways once infiltration has occurred. Animpediment to the success of combination therapies, however, is thatthey traditionally require administration of at least three differentdrug products (a vaccine, a T cell costimulatory, and a checkpointinhibitor), each of which has a significant cost and, in some cases,toxicity.

This document is based, at least in part, on the discovery that acombination of a vaccination, e.g. gp96-Ig vaccination, and T cellcostimulation with one or more agonists of OX40, ICOS, 4-1BB, TNFRSF25,CD40, CD27, and/or GITR, among others, provides a synergistic anti-tumorbenefit. Pre-clinical models have evaluated independent compositions ofgp96-Ig vaccines combined with agonistic antibodies targeting OX40,ICOS, 4-1BB, and TNFRSF25, and demonstrated variable effects onmechanistic and anti-tumor complementarity. The materials and methodsdescribed herein are advantageous in that, inter alia, they provide asingle composition that can achieve both vaccination with, for example,gp96-Ig, and T cell costimulation without the need for independentproducts. These materials and methods achieve this goal by creating asingle vaccine protein (e.g., gp96-Ig) expression vector that has beengenetically modified to simultaneously express an costimulatorymolecule, including without limitation, fusion proteins such asICOSL-Ig, 4-1BBL-Ig, TL1A-Ig, OX40L-Ig, CD40L-Ig, CD70-Ig, or GITRL-Igto provide T cell costimulation. The vectors, and methods for their use,can provide a costimulatory benefit without the need for an additionalantibody therapy to enhance the activation of antigen-specific CD8+ Tcells. Thus, combination immunotherapy can be achieved by vectorre-engineering to obviate the need for vaccine/antibody/fusion proteinregimens, which may reduce both the cost of therapy and the risk ofsystemic toxicity.

In one aspect, this document features an expression vector containing afirst nucleotide sequence that encodes a secretable vaccine protein, anda second nucleotide sequence that encodes a T cell costimulatory fusionprotein, wherein the T cell costimulatory fusion protein enhancesactivation of antigen-specific T cells when administered to a subject.In some embodiments, this document features an expression vectorcontaining a first nucleotide sequence that encodes a secretable gp96-Igfusion protein, and a second nucleotide sequence that encodes a T cellcostimulatory fusion protein, wherein the T cell costimulatory fusionprotein enhances activation of antigen-specific T cells whenadministered to a subject. The expression vector can be a mammalianexpression vector. In an embodiment, the secretable gp96-Ig fusionprotein can lack the gp96 KDEL (SEQ ID NO:3) sequence. The Ig tag in thegp96-Ig fusion protein can include the Fc region of human IgG1, IgG2,IgG3, IgG4, IgM, IgA, or IgE. The T cell costimulatory fusion proteincan be OX40L-Ig or a portion thereof that binds to OX40, ICOSL-Ig or aportion thereof that binds to ICOS, 4-1BBL-Ig or a portion thereof thatbinds to 4-1BBR, TL1A-Ig or a portion thereof that binds to TNFRSF25,GITRL-Ig or a portion thereof that binds to GITR, CD40-Ig or a portionthereof that binds to CD40, or CD70-Ig or a portion thereof that bindsto CD27, among others. The Ig tag in the T cell costimulatory fusionprotein can include the Fc region of human IgG1, IgG2, IgG3, IgG4, IgM,IgA, or IgE. The expression vector can contain DNA or RNA.

In another aspect, this document features a composition containing anexpression vector that comprises a first nucleotide sequence encoding asecretable vaccine protein, such as a secretable gp96-Ig fusion protein,and a second nucleotide sequence encoding a T cell costimulatory fusionprotein, wherein the T cell costimulatory fusion protein enhancesactivation of antigen-specific T cells when administered to a subject.The vector can be a DNA-based mammalian expression vector. In anembodiment, the secretable gp96-Ig fusion protein can lack the gp96 KDEL(SEQ ID NO:3) sequence. The Ig tag in the gp96-Ig fusion protein cancontain the Fc region of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE.The T cell costimulatory fusion protein can be OX40L-Ig or a portionthereof that binds to OX40, ICOSL-Ig or a portion thereof that binds toICOS, 4-1BBL-Ig or a portion thereof that binds to 4-1BBR, TL1A-Ig or aportion thereof that binds to TNFRSF25, GITRL-Ig or a portion thereofthat binds to GITR, CD40L-Ig or a portion thereof that binds to CD40, orCD70-Ig or a portion thereof that binds to CD27. The Ig tag in the Tcell costimulatory fusion protein can include the Fc region of humanIgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE. The expression vector can beincorporated into a virus or virus-like particle, or can be incorporatedinto a human tumor cell (e.g., a human tumor cell from an establishedcell line, e.g. a NSCLC, bladder cancer, melanoma, ovarian cancer, renalcell carcinoma, prostate carcinoma, sarcoma, breast carcinoma, squamouscell carcinoma, head and neck carcinoma, hepatocellular carcinoma,pancreatic carcinoma, or colon carcinoma cell line).

In another aspect, this document features a cell comprising acomposition containing an expression vector that comprises a firstnucleotide sequence encoding a secretable vaccine protein, and a secondnucleotide sequence encoding a T cell costimulatory fusion protein,wherein the T cell costimulatory fusion protein enhances activation ofantigen-specific T cells when administered to a subject. In someembodiments, this document features a cell comprising a compositioncontaining an expression vector that comprises a first nucleotidesequence encoding a secretable gp96-Ig fusion protein, and a secondnucleotide sequence encoding a T cell costimulatory fusion protein,wherein the T cell costimulatory fusion protein enhances activation ofantigen-specific T cells when administered to a subject. Such a cell, invarious embodiments, can be suitable for use as an off-the-shelftherapy. Such a cell, in various embodiments, is irradiated. Such acell, in various embodiments, is live and attenuated. These cells, invarious embodiments, express tumor antigens which may be chaperoned bythe vaccine protein (e.g., gp96) of the present compositions. Such acell, in various embodiments, can be derived from an established cellline e.g., a human tumor cell from an established NSCLC, bladder cancer,melanoma, ovarian cancer, renal cell carcinoma, prostate carcinoma,sarcoma, breast carcinoma, squamous cell carcinoma, head and neckcarcinoma, hepatocellular carcinoma, pancreatic carcinoma, or coloncarcinoma cell line. Such a cell, in various embodiments, can be derivedfrom an established prostate cancer cell line. Such a cell, in variousembodiments, can be derived from an established lung cancer cell line.Such a cell, in various embodiments, can be derived from an establishedbladder cancer cell line. Such a cell, in various embodiments, can bederived from an established sarcoma cell line. Such a cell, in variousembodiments, can be derived from an established choriocarcinoma cancercell line.

In another aspect, this document features a method for treating asubject. The method can include administering to a subject an effectiveamount of a composition described herein, for instance, containing anexpression vector that includes a first nucleotide sequence encoding asecretable vaccine protein such as a secretable gp96-Ig fusion protein,and a second nucleotide sequence encoding a T cell costimulatory fusionprotein, wherein the T cell costimulatory fusion protein enhancesactivation of antigen-specific T cells when administered to the subject.The vector can be incorporated into a virus or virus-like particle, orincorporated into a human tumor cell. The subject can be a human cancerpatient. Administration of the composition to the human patient canincrease the activation or proliferation of tumor antigen specific Tcells in the patient. For example, the activation or proliferation oftumor antigen specific T cells in the patient can be increased by atleast 25 percent (e.g., at least 30 percent, at least 40 percent, atleast 50 percent, at least 60 percent, at least 70 percent, or at least75 percent) as compared to the level of activation or proliferation oftumor antigen specific T cells in the patient prior to theadministration. The method can include administering the composition toa human cancer patient in combination with an agent that inhibitsimmunosuppressive molecules produced by tumor cells. The agent can be anantibody against PD-1. The subject can be a human with an acute orchronic infection (e.g., an infection by hepatitis C virus, hepatitis Bvirus, human immunodeficiency virus, or malaria). Administration of thecomposition to the human patient can stimulate the activation orproliferation of pathogenic antigen specific T cells. The T cellcostimulatory molecule can enhance the activation of antigen-specific Tcells in the subject to a greater level than gp96-Ig vaccination alone.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of the re-engineering of anoriginal gp96-Ig vector to generate a cell-based combination productthat encodes the 96-Ig fusion protein in a first cassette, and a T cellcostimulatory fusion protein in a second cassette. ICOS-Fc, 4-1BBL-Fc,and OX40L-Fc are shown for illustration.

FIG. 2 is a schematic representation of a mammalian expression vector(B45) encoding a secretable gp96-Ig fusion protein in one expressioncassette and a T cell costimulatory fusion protein (by way ofnon-limiting illustration, ICOSL-IgG4 Fc) in a second cassette.

FIG. 3 is an illustration of an allogeneic tumor cell that has beentransfected with a vector encoding two secretable proteins. The firstprotein, gp96-Ig, forms a secretable dimer (smooth) that chaperonescell-derived antigens outside the cells. The second protein is atrimeric secretable T cell costimulatory fusion protein (rough) which issecreted by the vaccine cell and may freely bind to a nearbycostimulatory receptor on the surface of a T cell.

FIGS. 4A-4G show that an OX40 agonist antibody in combination with 96-Igcellular vaccine promotes antigen specific CD8 proliferation, whileFOXP3+Tregs remain unaffected. FIGS. 4A-4D depict the gp96-Ig cellularvaccine mechanism of action. In FIG. 4A, vaccine cells secrete gp96-Igalong with cell-derived antigens, or in the case of FIGS. 5, 6, and 8,the single antigen chicken ovalbumin which is stably expressed in thisvaccine cell line. In FIG. 4B, gp96-Ig/antigen complexes are taken up byAPCs and the antigens are transferred to MHC class I molecules. In FIG.4C, antigen cross-presentation leads to CD8+ specific T cell activation.In FIG. 4D, in the context of a tumor, the activated CD8+ T cells canrecognize shared tumor antigens on distant tumors and destroy them. FIG.4E is a graph plotting antigen specific (OT-1) cell expansion followingvaccination with ImPACT (as used herein, this refers to a modified (e.g.KDEL deletion) gp96-Ig fusion protein or, in some cases, an engineeredcell line designed to express the gp96-Ig fusion protein) alone or incombination with agonistic T cell co-stimulatory antibodies for ICOS,4-1BB, or OX40. Data are shown for days 5 and 40 following the initialvaccination (prime and memory, respectively). The latter alsocorresponds to 5 days after the second, boost vaccination. Only theImPACT/OX40(ab) combination generated OT-1 expansion that wassignificantly greater than ImPACT on its own (*, p<.05). Experimentalreplicates are listed. Plotted values are the mean and error is SEM. Foreach data set in FIG. 4E, the order of histograms from left to right is:No Vaccine, ImPACT alone, ImPACT plus ICOS antibody, ImPact plus OX40antibody, and ImPact plus 4-1BB antibody. FIG. 4F is a graph plottingFOXP3+ Tregs as a percentage of total CD4+ cells. With the exception ofImPACT/4-1BB treated mice, which fluctuated from increased to decreasedFOXP3+ cells, there was no significant change in Tregs following ImPACTtreatment, emphasizing its specificity towards CD8+ expansion.Experimental replicates are listed. Plotted values are the mean anderror is SEM. For each data set in FIG. 4F, the order of histograms fromleft to right is: No Vaccine, ImPACT alone, ImPact plus ICOS antibody,ImPact plus OX40 antibody, and ImPact plus 4-1BB antibody. FIG. 4G is apair of graphs plotting OT-1/CD8 and FOXP3+ cell expansion in a secondmodel system in response to the adjuvant Alum. Again, the combination ofImPACT and OX40(ab) resulted in significant OT-1 proliferation, inaddition to a moderate increase in FOXP3+ cells. Experimental replicatesare listed. Plotted values are the mean and error is SEM. For each dataset in FIG. 4G, the order of histograms from left to right is: NoVaccine, IgG control, and ImPact plus OX40 antibody.

FIGS. 5A-5D show that T cell co-stimulator OX40 functionssynergistically with the ImPACT (gp96-Ig) cellular vaccine to activate Tcells and produce antigen specific CD8+ expansion. FIG. 5A is aschematic representation of receptor (OX40, ICOS, and 4-1BB) and ligand(OX40L, ICOSL, and 4-1BBL) interactions between T cells and antigenpresenting cells (APC) promoting T cell activation. FIG. 5B is a diagramdepicting a vaccine cell line established through selection of a clonalpopulation of cells expressing gp96-Ig/HLA-A1 (ImPACT) along with thesingle antigen chicken ovalbumin, in order to track antigen specific Tcell expansion (OT-I cells) following the administration of vaccine.FIG. 5C is a list of murine T cell co-stimulator agonist antibodiestested in combination with ImPACT for synergism in promoting T cellexpansion—all of these antibodies are useful in various embodiments ascombination therapy agents. FIG. 5D is a graph plotting OT-1 levels inFOXP3-RFP reporter mice that were seeded with antigen specific OT-1(CD8) cells labeled with GFP via tail vein injection on day −1, asdetected by flow cytometry for 43 days after vaccination with eitherImPACT alone, or ImPACT in combination with 100 μg of agonist T cellco-stimulatory antibodies for OX40, ICOS, or 4-1BB on day 0.Unvaccinated (No Vaccine) mice were assessed in parallel as a control.Mice were boosted with vaccine or vaccine/antibody combinations again onday 35. Vaccination days are indicated by syringes. The initial (prime)response peaked on day 5, and only vaccine/OX40(ab) treated mice showeda modest memory response following boost (arrows). Plotted valuesrepresent the mean percentage of OT-1 cells from all CD8+ cells anderror is SEM. See also FIG. 4A-G for the number of experimentalreplicates and statistical significance between sample groups.

FIGS. 6A-6C show that the combination of T cell co-stimulator OX40L withImPACT into a new vaccine vector (“ComPACT”) produced surprisinglysuperior antigen specific CD8+ T cell expansion as compared tocoadministration of OX40 agonist antibody. FIG. 6A depicts theexperimental design to compare (a) antigen specific T cell expansionusing the original vaccine ImPACT (Gp96-Ig) in combination with OX40agonist antibody to (b) the new vaccine ComPACT (in this figure,Gp96-Ig/OX40L-Fc). FIG. 6B depicts the peak of antigen-specific CD8+ Tcell proliferation following primary immunization with a controlvaccine, a vaccine expressing ovalbumin, a vaccine expressing ovalbuminand gp96-Ig (ImPACT), ImPACT in combination with OX40 agonistantibodies, or ComPACT. For each data set in FIG. 6B, the order of peaksfrom left to right is: No Vaccine, Ova only control, ImPACT, ImPACT plusOX40 antibody, and ComPACT. FIG. 6C is a graph plotting the OT-1expansion time-course (similar to FIG. 5D), using FOXP3-RFP mice seededwith OT-1 (CD8) cells on day −1. OT-1/GFP cells were analyzed by flowcytometry in mice treated with No Vaccine, Ova only control cells,ImPACT, ImPACT+100 μg OX40(ab), and ComPACT, over 46 days, with initialvaccination on day 0 and a boost on day 35 (indicated by syringes). Bothprime and memory responses (arrows) were greatest in mice treated withComPACT, even when compared with ImPACT+OX40(ab). ComPACT mice alsosurprisingly retained elevated OT-1 levels throughout the time-course(˜days 7-20). Plotted values represent the mean and error is SEM. Seealso FIGS. 7A-7F for the number of experimental replicates andstatistical significance between sample groups.

FIGS. 7A-7F show that the combination of gp96-Ig and OX40L, ICOSL, or4-1BBL expression in ComPACT results in high-level CD8, antigen specificT cell response. FIGS. 7A-7D show the characterization of the3T3-version of ComPACT as used in FIG. 6C. 3T3 cells were transfectedwith a plasmid expressing chicken ovalbumin (Ova), and a singlehigh-expressing clone was established and used to re-transfect withVaccine vectors (either 96-Ig alone, gp96-Ig/OX40L-Fc, gp96-Ig/ICOSL, orgp96-Ig/4-1BBL). Vaccines were therefore established in the same Ovaparental clone. Unvaccinated mice (No vaccine) were compared with micetreated with Ova only expressing cells (as an additional control),ImPACT (Ova-gp96-Ig), ImPACT+OX40 agonist antibody (OX40(ab)), ComPACT(Ova-gp96-Ig/ICOSL), ComPACT (Ova-gp96-Ig/OX40L-Fc), or ComPACT(Ova-gp96-Ig/1BBL). FIG. 7A is a graph plotting Ova secretion asconfirmed by ELISA, showing that secretion was essentially identicalbetween Ova only control, ImPACT, and the various ComPACT cells. Valuesare the mean from a minimum of 6 replicates and error is SEM. For eachdata set in FIG. 7A, the order of histograms from left to right is: Ovaonly control, ImPACT (3T3-ova-gp96-Ig), ComPACT (Ova-gp96-Ig/ICOSL),ComPACT (Ova-gp96-Ig/OX40L-Fc), and ComPACT (Ova-gp96-Ig/1BBL). FIG. 7Bis a graph plotting gp96-Ig secretion (detected as IgG) as determined byELISA, showing that individual ImPACT and ComPACT clones wereestablished that secreted comparable levels. Values are the mean from aminimum of 6 replicates and error is SEM. For each data set in FIG. 7B,the order of histograms from left to right is: Ova only control, ImPACT(3T3-ova-gp96-Ig), ComPACT (Ova-gp96-Ig/ICOSL), ComPACT(Ova-gp96-Ig/OX40L-Fc), and ComPACT (Ova-gp96-Ig/1BBL). FIG. 7C is agraph plotting mRNA expression of ICOSL, OX40L, or 4-1BBL as confirmedby qRT-PCR, showing expression only in ComPACT cells. Graphical valuesare the mean from a minimum of 3 distinct replicates and error is SEM.For each data set in FIG. 7C, the order of histograms from left to rightis: Ova only control, ImPACT (3T3-ova-gp96-Ig), ComPACT(Ova-gp96-Ig/ICOSL), ComPACT (Ova-gp96-Ig/OX40L-Fc), and ComPACT(Ova-gp96-Ig/1BBL). FIG. 7D provides Western blots showing confirmationof OX40L, ICOSL, and 4-1BBL expression in ComPACT cells. ImPACT andComPACT cells were treated with Brefeldin A (BFA) for 16 hours toprevent protein transport and secretion. Cells were then harvested,lysed and subjected to SDS PAGE/Western blot analysis. Blots were probedwith an antibody to OX40L (also known as CD252), ICOSL, or 4-1BBL, andhistone H3 or actin B (ACTB) as a loading control. OX40L, ICOSL, and4-1BBL were only detected in ComPACT cells. FIG. 7E is a graph plottingthe frequency of CD4+FoxP3+ regulatory T cells on day 5 following theindicated primary immunization. For each data set in FIG. 7E, the orderof peaks from left to right is: No vaccine, Ova only control, ImPACT,ImPACT plus OX40 antibody, and ComPACT (in this figure,Ova-gp96-Ig/OX40L-Fc). FIG. 7F is a pair of graphs plotting thefrequency of antigen-specific CD8+ T cells (OT-I) in the peripheralblood on day 42 (7 days following the boost immunization), shown on theleft, and the peak of CD4+FoxP3+ T regulatory cells on the same day inthe peripheral blood. As in FIG. 6C, antigen specific (OT-1) cellexpansion following vaccination with an Ova only expressing cell line,ImPACT, ImPACT in combination with OX40(ab) and ComPACT (in this case,Ova-gp96-Ig/OX40L-Fc), are shown at 5 and 40 days following the initialvaccination (prime and memory, respectively). The latter alsocorresponds to 5 days after the second, boost vaccination. OT-1 levelsin ImPACT, ImPACT+OX40(ab) and ComPACT treated mice are significantlyelevated compared to Ova only control treated mice. ComPACT treated miceexhibit the greatest proliferation of OT-1 cells, which aresignificantly higher than ImPACT+OX40(ab) at both prime and memoryresponse points. Experimental replicates are listed and error is SEM.For each data set in FIG. 7F, the order of peaks from left to right is:No vaccine, Ova only control, ImPACT, ImPACT plus OX40 antibody, andComPACT (in this figure, Ova-gp96-Ig/OX40L-Fc).

FIGS. 8A-8E show that ComPACT elicited antigen specific CD8+ expansionwhile OX40 antibody led to non-specific T cell activation. FIG. 8A is aseries of graphs plotting total numbers of mononuclear (MNC), CD4, CD8,OT-I, and OT-II cells in mice that were either untreated or vaccinatedwith ImPACT, ImPACT+OX40(ab), or ComPACT (in this figure,Gp96-Ig/OX40L-Fc). As for FIGS. 5D and 6C, FOXP3-RFP reporter mice wereseeded with OT-1 cells via tail vein injection on day −1, vaccinated onday 0, and sacrificed on day 8 for analysis, including flow cytometry ofcells obtained from peritoneal wash. ComPACT treatment produced a robustOT-I (CD8) specific response, whereas OX40(ab) treatment resulted in anincrease in all T cell sub-types, including FOXP3+ CD4 cells. Plottedvalues represent the mean from a minimum of 3 mice and error is SEM. Foreach data set in FIG. 8A, the order of peaks from left to right is:Untreated, ImPACT, ImPACT plus OX40 antibody, and ComPACT (in thisfigure, Gp96-Ig/OX40L-Fc). FIG. 8B is a series of graphs plottingnumbers of CD127⁺KLRG1⁻, CD127⁻KLRG1⁺, and CD127⁺KLRG1⁻ cells, whichcorrespond to memory precursor cells, short-lived effector cells, andmemory cells, respectively, on day 8 following the primary immunization.The cells were derived from the spleen (top panels) and peritonealcavity (bottom panels). For each data set in FIG. 8B, the order of peaksfrom left to right is: Untreated, Ova only, ImPACT, ComPACT(Ova-gp96-Ig/ICOSL), ComPACT (Ova-gp96-Ig/OX40L-Fc), and ComPACT(Ova-gp96-Ig/1BBL). FIG. 8C is a series of graphs plotting levels ofINFγ, TNFα, IL2, IL6, and IL5. Whole blood serum was harvested from thesame mice presented in FIG. 8A above on day 8, and subjected to cytokineanalysis using the LEGENDPLEX™ kit from BioLegend and flow cytometer.Consistent with the data of FIG. 8A, OX40(ab) treatment produced anon-specific, systemic immune response, with elevated levels of not onlyINFγ, TNFα and IL2, but also IL6 and IL5. Plotted values represent themean from a minimum of 3 mice and error is SEM. For each data set inFIG. 8C, the order of peaks from left to right is: Untreated, ImPACT,ImPACT plus OX40 antibody, and ComPACT (in this figure,Gp96-Ig/OX40L-Fc). FIG. 8D is a series of graphs plotting geneexpression levels for FNγ, TNFα, and Granzyme-B (GZMB). Analysis of Tcell activation genes by qRT-PCR demonstrated ComPACT's specificity inonly activating antigen specific CD8 (OT-I+) cells, compared toOX40(ab), which non-specifically activated both endogenous (OT-I−) andantigen specific CD8 (OT-I+) cells. Cells obtained from peritonealwashes in FIG. 8A above were sorted into populations of OT-1− and OT-1+CD8 cells. Total RNA was harvested, reverse transcribed and analyzed byqPCR. Gene expression levels of IFNγ, TNFα, and GZMB are shown,normalized to 18S mRNA with the first ImPACT only treated replicate setat 1. Plotted values represent the mean from a minimum of 3 mice, anderror is SEM. For each data set in FIG. 8D, the order of histograms fromleft to right is: ImPACT, ImPACT plus OX40 antibody, and ComPACT (inthis figure, Gp96-Ig/OX40L-Fc). FIG. 8E shows the number of FOXP3regulatory T cells (Treg) in splenocytes and tumor draining lymph node(TDLN) in the mice. For each data set in FIG. 8E, the order of peaksfrom left to right is: Untreated, ImPACT, ImPACT plus OX40 antibody, andComPACT (in this figure, Gp96-Ig/OX40L-Fc).

FIGS. 9A-9C show that ComPACT (in this figure, Gp96-Ig/OX40L-Fc)treatment results in antigen specific CD8 T cell activation, whereascoadministration of OX40(ab) treatment elicits non-specific immune cellactivation including increases in FOXP3 Tregs in both the spleen andlymph nodes. FIG. 9A is a series of graphs plotting total numbers ofMNC, CD4, CD8, OT-I, and OT-II cells for mice either untreated orvaccinated with ImPACT, ImPACT+OX40(ab), or ComPACT. As in FIGS. 5D and6C, FIR reporter mice were seeded with OT-1 cells via tail veininjection on day −1, vaccinated on day 0, and sacrificed on day 8 foranalysis, including flow cytometry of cells obtained from the spleen.OX40(ab) treated mice demonstrated an increase in all T cell sub-types,including CD4/FOXP3+ cells. ComPACT treated mice produced a robust OT-1(CD8) specific response that was significantly higher than the OX40(ab)response. Plotted values represent the mean from a minimum of 3 mice anderror is SEM. For each data set in FIG. 9A, the order of peaks from leftto right is: Untreated, ImPACT, ImPACT plus OX40 antibody, and ComPACT.FIG. 9B is a series of graphs plotting total numbers of MNC, CD4, CD8,OT-I, and OT-II cells for mice either untreated or vaccinated withImPACT, ImPACT+OX40(ab), or ComPACT as in FIG. 9A, except in peripherallymph nodes. For each data set in FIG. 9B, the order of peaks from leftto right is: Untreated, ImPACT, ImPACT plus OX40 antibody, and ComPACT.FIG. 9C is a series of graphs plotting mRNA expression for T cellactivation genes (ACTB, IL2, and Perforin 1 (PRF1)). qRT-PCR revealedantigen specific OT-1 (CD8) activation in mice treated with ComPACT,compared to non-specific activation of both endogenous and antigenspecific OT-1 CD8 cells in mice treated with OX40(ab). Cells obtainedfrom peritoneal washes in FIG. 8A above were sorted into populations ofOT-1⁺ and OT-1⁻CD8 cells. Total RNA was harvested, reverse transcribed,and analyzed by qPCR. ACTB levels were consistent between cellpopulations and treatments, serving as a control. IL2 levels weresignificantly elevated in OT-1⁺ cells of mice treated with ImPACT,ImPACT+OX40(ab), and ComPACT, indicating significant T cell activationwith all vaccines/combinations. Consistent with FIG. 8C, levels of PRF1were elevated non-specifically in both OT-1⁻ and OT-1⁺CD8 fractions ofmice treated with OX40(ab), while only increasing in OT-1⁺ cells ofComPACT treated mice. Plotted values represent the mean from a minimumof 3 mice and error is SEM. For each data set in FIG. 9C, the order ofhistograms from left to right is: ImPACT, ImPACT plus OX40 antibody, andComPACT.

FIGS. 10A-10C show that in tumor bearing mice, ComPACT (in this figure,Gp96-Ig/OX40L-Fc) treatment resulted in the maximum number of tumorinvading lymphocytes and tumor regression. FIG. 10A is a schematic ofthe experimental setup. BALB/C mice were inoculated with 2×10⁵ CT26cells sub-dermally, indicating day 0. On days 6 and 11, mice were eitherunvaccinated or vaccinated with ImPACT, ImPACT+OX86(ab), ComPACT, orOX86(ab) alone. Vaccine treatments consisted of 1×10⁶ cells and 100 μgof antibody. FIG. 10B is a graph plotting tumor area on the indicateddays following tumor inoculation on day 0 and is plotted as the meanfrom a minimum of 5 experimental mice per sample group, with error asSEM. FIG. 10C is a graph plotting tumor area on day 21 of the study. Foreach data set in FIG. 10C, the order of peaks from left to right is: Novaccine, CT26 only control, OX40 antibody only, ImPACT, ImPACT plus OX40antibody, and ComPACT.

FIGS. 11A-11E show that ComPACT (in this figure, Gp96-Ig/OX40L-Fc)treatment resulted in CD8+ specific tumor infiltration, hindered tumorgrowth, increased overall survival and significant tumor rejection inthe CT26 colorectal carcinoma model. In FIG. 11A, mice were inoculatedon day 0 with 5×10⁵ CT26 tumor cells injected subcutaneously in the rearflank. Mice were either untreated or vaccinated on days 4, 7 and 10 withCT26 parental cells, OX40(ab) alone, ImPACT alone, ImPACT+OX40(ab) orComPACT. A cohort of mice were sacrificed on day 12 for tumor geneticanalysis. Remaining mice were monitored for 30 days to measure tumorarea and overall survival. FIG. 11B depicts analysis of day 12 tumorgene expression. Total RNA was isolated from dissociated tumors, reversetranscribed and analyzed by qPCR. Values were normalized to 18S mRNA andthe first ‘Untreated’ only replicate was set at 1. For each data set inFIG. 11B, the order of histograms from left to right is: Untreated, CT26only control, OX40 antibody only, ImPACT, ImPACT plus OX40 antibody, andComPACT. In FIG. 11C, AH1-tetramer/antigen specific CD8+ cells wereanalyzed in treated mice. For each data set in FIG. 11C, the order ofpeaks from left to right is: Untreated, CT26 only control, OX40 antibodyonly, ImPACT, ImPACT plus OX40 antibody, and ComPACT. FIG. 11D showstumor area as measured daily for 21 days following initial tumorinoculation. In FIG. 11E, overall survival was determined over a 30 daytime course. 80% of ComPACT treated mice survived according toexperimental criteria and 47% of mice (7 out of 15) completely rejectedestablished tumors. One OX40(ab) only treated mouse rejected the tumorby day 24 and one ImPACT+OX40(ab) treated mouse rejected by day 25.

FIGS. 12A-12D show that ComPACT (in this figure, Gp96-Ig/OX40L-Fc)generates antigen-specific CD8+ expansion, delayed tumor growth,increased overall survival and tumor rejection in an aggressiveB16.F10-ova melanoma model. In FIG. 12A, mice were adoptivelytransferred with 5×10⁵ OT-I cells on day −1, and then inoculated on day0 with 5×10⁵ B16.F10-ova tumor cells injected subcutaneously in the rearflank. Mice were either untreated or vaccinated on days 4, 7 and 10 withB16.F10-ova parental cells, OX40(ab) alone, ImPACT alone,ImPACT+OX40(ab) or ComPACT. FIG. 12B shows antigen-specific CD8+ (OT-I)expansion following treatment over a time-course of 25 days. In FIG.12C, tumor area was measured throughout a 25 day time course followinginitial tumor inoculation. In FIG. 12D, overall survival was determinedover a 30 day time course. Approximately 78% of ComPACT treated micesurvived and 11% of the ComPACT treated mice completely rejectedestablished tumors. Only the ComPACT treated group had complete tumorrejecters: 1 out of 9 mice or approximately 11%.

FIG. 13 is a graph plotting the OT-1 expansion time-course (similar toFIGS. 5D and 6C), using FOXP3-RFP mice seeded with OT-1 (CD8) cells onday −1. OT-1/GFP cells were analyzed by flow cytometry in mice treatedwith No Vaccine, Ova only control cells, ComPACT (96-Ig/OX40L orgp96-Ig/TL1A) or ComPACT² (96-Ig/OX40L+TL1A), over 46 days, with initialvaccination on day 0 and a boost on day 35. Plotted values represent themean and error is SEM. ComPACT² (gp96-Ig/OX40L+TL1A) represents acombination injection including ComPACT-OX40L and ComPACT-TL1A (i.e.,two different cell lines in the same syringe).

FIG. 14 is a graph showing the effects of ComPACT on the proliferationand activation of ovalbumin specific CD8+ T cells (OTI). C57BL/6 micewere immunized with ImPACT alone or ComPACT (gp96-Ig/OX40L), ComPACT(gp96-Ig/4-ICOSL), or ComPACT (gp96-Ig/4-1BBL) at day 0. The frequencyof OT-I was monitored in the peripheral blood on the indicated days.

FIG. 15 is a graph showing the effect of ComPACT on tumor growthkinetics in the CT26 colorectal carcinoma model. Mice were inoculated onday 0 with 5×10⁵ CT26 tumor cells injected subcutaneously in the rearflank. Mice were either untreated or vaccinated on days 4, 7 and 10 withCT26 parental cells, ImPACT alone, ImPACT+the TNFRSF25 agonist (4C12ab), 4C12 (ab) alone, PD-1 (ab) alone, 4C12 (ab) and PD-1 (ab), ComPACT(gp96-Ig/OX40L or gp96-Ig/TL1A), ComPACT (gp96-Ig/OX40L)+PD-1 (ab), orComPACT² (gp96-Ig/OX40L+TL1A). The mice were monitored for 30 days tomeasure tumor area. ComPACT² (gp96-Ig/OX40L+TL1A) represents acombination injection including ComPACT-OX40L and ComPACT-TL1A (i.e.,two different cell lines in the same syringe).

FIG. 16 is a graph showing the effect of ComPACT on overall micesurvival in the CT26 colorectal carcinoma model. Mice were treated withCT26 tumor cells and vaccinated as described in FIG. 15.

FIG. 17 is a graph showing the amount of human OX40L produced by a humanprostate specific vaccine (HS-1020, PC-3 cell line).

FIG. 18 is a graph showing the amount of human OX40L produced by a humanlung specific vaccine (HS-120, AD100 cell line).

DETAILED DESCRIPTION

Various secretable proteins, i.e. vaccine proteins as described herein,can be used to stimulate an immune response in vivo. For example,secretable heat-shock protein gp96-Ig based allogeneic cellular vaccinescan achieve high-frequency polyclonal CD8+ T cell responses tofemto-molar concentrations of tumor antigens through antigencross-priming in vivo (Oizumi et al., J Immunol 2007, 179(4):2310-2317).Multiple immunosuppressive mechanisms elaborated by established tumorscan dampen the activity of this vaccine approach, however. To evaluatethe potential utility of combination immunotherapy for patients withadvanced disease, a systematic comparison of PD-1, PD-L1, CTLA-4, andLAG-3 blocking antibodies in mouse models of long-established B16-F10melanoma was carried out (see, the Examples herein), demonstratingsuperior combination between gp96-Ig vaccination and PD-1 blockade ascompared to other checkpoints. Synergistic anti-tumor benefits mayresult from triple combinations of gp96-Ig vaccination, PD-1 blockade,and T cell costimulation using one or of an agonist of OX40 (e.g., anOX40 ligand-Ig (OX40L-Ig) fusion, or a fragment thereof that bindsOX40), an agonist of inducible T-cell costimulator (ICOS) (e.g., an ICOSligand-Ig (ICOSL-Ig) fusion, or a fragment thereof that binds ICOS), anagonist of CD40 (e.g., a CD40L-Ig fusion protein, or fragment thereof),an agonist of CD27 (e.g. a CD70-Ig fusion protein or fragment thereof),an agonist of 4-1BB (e.g., a 4-1BB ligand-Ig (4-1BBL-Ig) fusion, or afragment thereof that binds 4-1BB), an agonist of TNFRSF25 (e.g., aTL1A-Ig fusion, or a fragment thereof that binds TNFRSF25), or anagonist of glucocorticoid-induced tumor necrosis factor receptor (GITR)(e.g., a GITR ligand-Ig (GITRL-Ig) fusion, or a fragment thereof thatbinds GITF). The enthusiasm for development of such triple combinationsis tempered by the anticipated cost of such therapies, however. Tocircumvent this issue, vaccine protein expressing vectors (e.g., gp96-Igexpressing vectors) were re-engineered to simultaneously express T cellcostimulatory protein (e.g., ICOSL-Ig, 4-1BBL-Ig, or OX40L-Ig), toprovide a costimulatory benefit without the need for an additionalantibody therapy. The re-engineered vectors are provided herein, as aremethods for their use. When gp96-Ig and these costimulatory fusionproteins were secreted by allogeneic cell lines, enhanced activation ofantigen-specific CD8+ T cells was observed (see, the Examples herein).Thus, combination immunotherapy can be achieved by vector re-engineeringto obviate the need for completely separate vaccine/antibody/fusionprotein regimens.

Vaccine Proteins

Vaccine proteins can induce immune responses that find use in thepresent invention. In various embodiments, the present inventionprovides expression vectors comprising a first nucleotide sequence thatencode a secretable vaccine protein and a second nucleotide sequencethat encode a T cell costimulatory fusion protein. Compositionscomprising the expression vectors of the present invention are alsoprovided. In various embodiments, such compositions are utilized inmethods of treating subjects to stimulate immune responses in thesubject including enhancing the activation of antigen-specific T cellsin the subject. The present compositions find use in the treatment ofvarious diseases including cancer.

The heat shock protein (hsp) gp96, localized in the endoplasmicreticulum (ER), serves as a chaperone for peptides on their way to MHCclass I and II molecules. Gp96 obtained from tumor cells and used as avaccine can induce specific tumor immunity, presumably through thetransport of tumor-specific peptides to antigen-presenting cells (APCs)(J Immunol 1999, 163(10):5178-5182). For example, gp96-associatedpeptides are cross-presented to CD8 cells by dendritic cells (DCs).

A vaccination system was developed for antitumor therapy by transfectinga gp96-Ig G1-Fc fusion protein into tumor cells, resulting in secretionof gp96-Ig in complex with chaperoned tumor peptides (see, J Immunother2008, 31(4):394-401, and references cited therein). Parenteraladministration of gp96-Ig secreting tumor cells triggers robust,antigen-specific CD8 cytotoxic T lymphocyte (CTL) expansion, combinedwith activation of the innate immune system. Tumor-secreted gp96 causesthe recruitment of DCs and natural killer (NK) cells to the site of gp96secretion, and mediates DC activation. Further, the endocytic uptake ofgp96 and its chaperoned peptides triggers peptide cross presentation viamajor MHC class I, as well as strong, cognate CD8 activation independentof CD4 cells.

The vectors provided herein contain a first nucleotide sequence thatencodes a gp96-Ig fusion protein. The coding region of human gp96 is2,412 bases in length (SEQ ID NO:1), and encodes an 803 amino acidprotein (SEQ ID NO:2) that includes a 21 amino acid signal peptide atthe amino terminus, a potential transmembrane region rich in hydrophobicresidues, and an ER retention peptide sequence at the carboxyl terminus(GENBANK® Accession No. X15187; see, Maki et al., Proc Natl Acad Sci USA1990, 87:5658-5562). The DNA and protein sequences of human gp96 follow:

(SEQ ID NO: 1) atgagggccctgtgggtgctgggcctctgctgcgtcctgctgaccttcgggtcggtcagagctgacgatgaagttgatgtggatggtacagtagaagaggatctgggtaaaagtagagaaggatcaaggacggatgatgaagtagtacagagagaggaagaagctattcagaggatggattaaatgcatcacaaataagagaacttagagagaagtcggaaaagtttgccttccaagccgaagttaacagaatgatgaaacttatcatcaattcattgtataaaaataaagagattttcctgagagaactgatttcaaatgcttctgatgctttagataagataaggctaatatcactgactgatgaaaatgctctactggaaatgaggaactaacagtcaaaattaagtgtgataaggagaagaacctgctgcatgtcacagacaccggtgtaggaatgaccagagaagagaggttaaaaaccttggtaccatagccaaatctgggacaagcgagatttaaacaaaatgactgaagcacaggaagatggccagtcaacttctgaattgattggccagtttggtgtcggtttctattccgccttccttgtagcagataaggttattgtcacttcaaaacacaacaacgatacccagcacatctgggagtctgactccaatgaattactgtaattgctgacccaagaggaaacactctaggacggggaacgacaattacccttgtcttaaaagaagaagcatctgattaccttgaattggatacaattaaaaatctcgtcaaaaaatattcacagttcataaactacctatttatgtatggagcagcaagactgaaactgttgaggagcccatggaggaagaagaagcagccaaagaagagaaagaagaatctgatgatgaagctgcagtagaggaagaagaagaagaaaagaaaccaaagactaaaaaagttgaaaaaactgtctgggactgggaacttatgaatgatatcaaaccaatatggcagagaccatcaaaagaagtagaagaagatgaatacaaagctttctacaaatcattacaaaggaaagtgatgaccccatggcttatattcactttactgctgaaggggaagttaccttcaaatcaattttatagtacccacatctgctccacgtggtctgtagacgaatatggatctaaaaagagcgattacattaagctctatgtgcgccgtgtattcatcacagacgacttccatgatatgatgcctaaatacctcaattagtcaagggtgtggtggactcagatgatctccccttgaatgtttcccgcgagactcttcagcaacataaactgcttaaggtgattaggaagaagcttgacgtaaaacgctggacatgatcaagaagattgctgatgataaatacaatgatactttttggaaagaatttggtaccaacatcaagcttggtgtgattgaagaccactcgaatcgaacacgtcttgctaaacttcttaggttccagtcttctcatcatccaactgacattactagcctagaccagtatgtggaaagaatgaaggaaaaacaagacaaaatctacttcatggctgggtccagcagaaaagaggctgaatcttctccatttgttgagcgacttctgaaaaagggctatgaagttatttacctcacagaacctgtggatgaatactgtattcaggcccttcccgaatttgatgggaagaggaccagaatgagccaaggaaggagtgaagttcgatgaaagtgagaaaactaaggagagtcgtgaagcagttgagaaagaatttgagcctctgctgaattggatgaaagataaagcccttaaggacaagattgaaaaggctgtggtgtctcagcgcctgacagaatctccgtgtgctttggtggccagccagtacggatggtctggcaacatggagagaatcatgaaagcacaagcgtaccaaacgggcaaggacatctctacaaattactatgcgagtcagaagaaaacatttgaaattaatcccagacacccgctgatcagagacatgcttcgacgaattaaggaagatgaagatgataaaacagttttggatcttgctgtggattgatgaaacagcaacgcttcggtcagggtatcttttaccagacactaaagcatatggagatagaatagaaagaatgcttcgcctcagtttgaacattgaccctgatgcaaaggtggaagaagagcccgaagaagaacctgaagagacagcagaagacacaacagaagacacagagcaagacgaagatgaagaaatggatgtgggaacagatgaagaagaagaaacagcaaaggaatctacagctgaaaaagatgaattgtaa (SEQ ID NO: 2)MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAEK DEL.

A nucleic acid encoding a gp96-Ig fusion sequence can be produced usingthe methods described in U.S. Pat. No. 8,685,384, which is incorporatedherein by reference in its entirety. In some embodiments, the gp96portion of a gp96-Ig fusion protein can contain all or a portion of awild type gp96 sequence (e.g., the human sequence set forth in SEQ IDNO:2). For example, a secretable gp96-Ig fusion protein can include thefirst 799 amino acids of SEQ ID NO:2, such that it lacks the C-terminalKDEL (SEQ ID NO:3) sequence. Alternatively, the gp96 portion of thefusion protein can have an amino acid sequence that contains one or moresubstitutions, deletions, or additions as compared to the first 799amino acids of the wild type gp96 sequence, such that it has at least90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) sequence identity to the wild type polypeptide.

As used throughout this disclosure, the percent sequence identitybetween a particular nucleic acid or amino acid sequence and a sequencereferenced by a particular sequence identification number is determinedas follows. First, a nucleic acid or amino acid sequence is compared tothe sequence set forth in a particular sequence identification numberusing the BLAST 2 Sequences (B12seq) program from the stand-aloneversion of BLASTZ containing BLASTN version 2.0.14 and BLASTP version2.0.14. This stand-alone version of BLASTZ can be obtained online atfr.com/blast or at ncbi.nlm.nih.gov. Instructions explaining how to usethe B12seq program can be found in the readme file accompanying BLASTZ.B12seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to −1; -r is set to 2; and all other options are left at theirdefault setting. For example, the following command can be used togenerate an output file containing a comparison between two sequences:C:\B12seq c:\seg1.txt-j c:\seg2.txt-p blastn-o c:\output.txt-q−1-r 2. Tocompare two amino acid sequences, the options of B12seq are set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); -p is setto blastp; -o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq c:\seq1.txt-jc:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence (e.g., SEQ ID NO:1), or by anarticulated length (e.g., 100 consecutive nucleotides or amino acidresidues from a sequence set forth in an identified sequence), followedby multiplying the resulting value by 100. For example, a nucleic acidsequence that has 2,200 matches when aligned with the sequence set forthin SEQ ID NO:1 is 91.2 percent identical to the sequence set forth inSEQ ID NO:1 (i.e., 2,000÷2,412×100=91.2). It is noted that the percentsequence identity value is rounded to the nearest tenth. For example,75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15,75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It also is notedthat the length value will always be an integer.

Thus, in some embodiments, the gp96 portion of nucleic acid encoding agp96-Ig fusion polypeptide can encode an amino acid sequence thatdiffers from the wild type gp96 polypeptide at one or more amino acidpositions, such that it contains one or more conservative substitutions,non-conservative substitutions, splice variants, isoforms, homologuesfrom other species, and polymorphisms.

As defined herein, a “conservative substitution” denotes the replacementof an amino acid residue by another, biologically similar, residue.Typically, biological similarity, as referred to above, reflectssubstitutions on the wild type sequence with conserved amino acids. Forexample, conservative amino acid substitutions would be expected to havelittle or no effect on biological activity, particularly if theyrepresent less than 10% of the total number of residues in thepolypeptide or protein. Conservative substitutions may be made, forinstance, on the basis of similarity in polarity, charge, size,solubility, hydrophobicity, hydrophilicity, and/or the amphipathicnature of the amino acid residues involved. The 20 naturally occurringamino acids can be grouped into the following six standard amino acidgroups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutralhydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic:His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro;and (6) aromatic: Trp, Tyr, Phe. Accordingly, conservative substitutionsmay be effected by exchanging an amino acid by another amino acid listedwithin the same group of the six standard amino acid groups shown above.For example, the exchange of Asp by Glu retains one negative charge inthe so modified polypeptide. In addition, glycine and proline may besubstituted for one another based on their ability to disrupt α-helices.Additional examples of conserved amino acid substitutions, include,without limitation, the substitution of one hydrophobic residue foranother, such as isoleucine, valine, leucine, or methionine, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine, and the like. The term “conservative substitution” alsoincludes the use of a substituted amino acid residue in place of anun-substituted parent amino acid residue, provided that antibodiesraised to the substituted polypeptide also immunoreact with theun-substituted polypeptide.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

Mutations may also be made to the nucleotide sequences of the presentfusion proteins by reference to the genetic code, including taking intoaccount codon degeneracy.

The Ig portion (“tag”) of a gp96-Ig fusion protein can contain, forexample, a non-variable portion of an immunoglobulin molecule (e.g., anIgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE molecule). Typically, suchportions contain at least functional CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain Fusions also can be made usingthe carboxyl terminus of the Fc portion of a constant domain, or aregion immediately amino-terminal to the CH1 of the heavy or lightchain. The Ig tag can be from a mammalian (e.g., human, mouse, monkey,or rat) immunoglobulin, but human immunoglobulin can be particularlyuseful when the 96-Ig fusion is intended for in vivo use for humans.

DNAs encoding immunoglobulin light or heavy chain constant regions areknown or readily available from cDNA libraries. See, for example, Adamset al., Biochemistry 1980, 19:2711-2719; Gough et al., Biochemistry 198019:2702-2710; Dolby et al., Proc Natl Acad Sci USA 1980, 77:6027-6031;Rice et al., Proc Natl Acad Sci USA 1982, 79:7862-7865; Falkner et al.,Nature 1982, 298:286-288; and Morrison et al., Ann Rev Immunol 1984,2:239-256. Since many immunological reagents and labeling systems areavailable for the detection of immunoglobulins, gp96-Ig fusion proteinscan readily be detected and quantified by a variety of immunologicaltechniques known in the art, such as enzyme-linked immunosorbent assay(ELISA), immunoprecipitation, and fluorescence activated cell sorting(FACS). Similarly, if the peptide tag is an epitope with readilyavailable antibodies, such reagents can be used with the techniquesmentioned above to detect, quantitate, and isolate 96-Ig fusions.

In various embodiments, the 96-Ig fusion protein and/or thecostimulatory molecule fusions, comprises a linker. In variousembodiments, the linker may be derived from naturally-occurringmulti-domain proteins or are empirical linkers as described, forexample, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen etal., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contentsof which are hereby incorporated by reference. In some embodiments, thelinker may be designed using linker designing databases and computerprograms such as those described in Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369 and Crasto et. al., (2000), Protein Eng.13(5):309-312, the entire contents of which are hereby incorporated byreference.

In some embodiments, the linker is a synthetic linker such as PEG.

In other embodiments, the linker is a polypeptide. In some embodiments,the linker is less than about 100 amino acids long. For example, thelinker may be less than about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids long. In some embodiments, the linker isflexible. In another embodiment, the linker is rigid. In variousembodiments, the linker is substantially comprised of glycine and serineresidues (e.g. about 30%, or about 40%, or about 50%, or about 60%, orabout 70%, or about 80%, or about 90%, or about 95%, or about 97%glycines and serines).

In various embodiments, the linker is a hinge region of an antibody(e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1,IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region, found inIgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer,allowing the Fab portion to move freely in space. In contrast to theconstant regions, the hinge domains are structurally diverse, varying inboth sequence and length among immunoglobulin classes and subclasses.For example, the length and flexibility of the hinge region varies amongthe IgG subclasses. The hinge region of IgG1 encompasses amino acids216-231 and, because it is freely flexible, the Fab fragments can rotateabout their axes of symmetry and move within a sphere centered at thefirst of two inter-heavy chain disulfide bridges. IgG2 has a shorterhinge than IgG1, with 12 amino acid residues and four disulfide bridges.The hinge region of IgG2 lacks a glycine residue, is relatively short,and contains a rigid poly-proline double helix, stabilized by extrainter-heavy chain disulfide bridges. These properties restrict theflexibility of the IgG2 molecule. IgG3 differs from the other subclassesby its unique extended hinge region (about four times as long as theIgG1 hinge), containing 62 amino acids (including 21 prolines and 11cysteines), forming an inflexible poly-proline double helix. In IgG3,the Fab fragments are relatively far away from the Fc fragment, givingthe molecule a greater flexibility. The elongated hinge in IgG3 is alsoresponsible for its higher molecular weight compared to the othersubclasses. The hinge region of IgG4 is shorter than that of IgG1 andits flexibility is intermediate between that of IgG1 and IgG2. Theflexibility of the hinge regions reportedly decreases in the orderIgG3>IgG1>IgG4>IgG2.

Additional illustrative linkers include, but are not limited to, linkershaving the sequence LE, GGGGS (SEQ ID NO:26), (GGGGS)_(n) (n=1-4) (SEQID NO: 27), (Gly)₈ (SEQ ID NO:28), (Gly)₆ (SEQ ID NO:29), (EAAAK)_(n)(n=1-3) (SEQ ID NO: 30), A(EAAAK)_(n)A (n=2-5) (SEQ ID NO: 31),AEAAAKEAAAKA (SEQ ID NO: 32), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 33),PAPAP (SEQ ID NO: 34), KESGSVSSEQLAQFRSLD (SEQ ID NO: 35),EGKSSGSGSESKST(SEQ ID NO: 36), GSAGSAAGSGEF (SEQ ID NO: 37), and(XP)_(n), with X designating any amino acid, e.g., Ala, Lys, or Glu.

In various embodiments, the linker may be functional. For example,without limitation, the linker may function to improve the foldingand/or stability, improve the expression, improve the pharmacokinetics,and/or improve the bioactivity of the present compositions. In anotherexample, the linker may function to target the compositions to aparticular cell type or location.

In some embodiments, a gp96 peptide can be fused to the hinge, CH2 andCH3 domains of murine IgG1 (Bowen et al., J Immunol 1996, 156:442-449).This region of the IgG1 molecule contains three cysteine residues thatnormally are involved in disulfide bonding with other cysteines in theIg molecule. Since none of the cysteines are required for the peptide tofunction as a tag, one or more of these cysteine residues can besubstituted by another amino acid residue, such as, for example, serine.

Various leader sequences known in the art also can be used for efficientsecretion of gp96-Ig fusion proteins from bacterial and mammalian cells(see, von Heijne, J Mol Biol 1985, 184:99-105). Leader peptides can beselected based on the intended host cell, and may include bacterial,yeast, viral, animal, and mammalian sequences. For example, the herpesvirus glycoprotein D leader peptide is suitable for use in a variety ofmammalian cells. Another leader peptide for use in mammalian cells canbe obtained from the V-J2-C region of the mouse immunoglobulin kappachain (Bernard et al., Proc Natl Acad Sci USA 1981, 78:5812-5816). DNAsequences encoding peptide tags or leader peptides are known or readilyavailable from libraries or commercial suppliers, and are suitable inthe fusion proteins described herein.

Furthermore, in various embodiments, one may substitute the gp96 of thepresent disclosure with one or more vaccine proteins. For instance,various heat shock proteins are among the vaccine proteins. In variousembodiments, the heat shock protein is one or more of a small hsp,hsp40, hsp60, hsp70, hsp90, and hsp110 family member, inclusive offragments, variants, mutants, derivatives or combinations thereof(Hickey, et al., 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol.Cell. Biol. 9:2279-2283).

T-Cell Co-Stimulation

In addition to a gp96-Ig fusion protein, the expression vectors providedherein can encode one or more biological response modifiers. In variousembodiments, the present expression vectors can encode one or more Tcell costimultory molecules.

In various embodiments, the present expression vectors allow for arobust, antigen-specific CD8 cytotoxic T lymphocyte (CTL) expansion. Invarious embodiments, the present expression vectors selectively enhanceCD8 cytotoxic T lymphocyte (CTL) and do not substantially enhance T celltypes that can be pro-tumor, and which include, but are not limited to,Tregs, CD4+ and/or CD8+ T cells expressing one or more checkpointinhibitory receptors, Th2 cells and Th17 cells. Checkpoint inhibitoryreceptors refers to receptors (e.g. CTLA-4, B7-H3, B7-H4, TIM-3)expressed on immune cells that prevent or inhibit uncontrolled immuneresponses. For instance, the present expression vectors do notsubstantially enhance FOXP3⁺ regulatory T cells. In some embodiments,this selective CD8 T cell enhancement is in contrast to the non-specificT cell enhancement observed with a combination therapy of a gp-96 fusionand an antibody against a T cell costimultory molecule.

For example, a vector can encode an agonist of OX40 (e.g., an OX40ligand-Ig (OX40L-Ig) fusion, or a fragment thereof that binds OX40), anagonist of inducible T-cell costimulator (ICOS) (e.g., an ICOS ligand-Ig(ICOSL-Ig) fusion, or a fragment thereof that binds ICOS), an agonist ofCD40 (e.g., a CD40L-Ig fusion protein, or fragment thereof), an agonistof CD27 (e.g. a CD70-Ig fusion protein or fragment thereof), or anagonist of 4-1BB (e.g., a 4-1BB ligand-Ig (4-1BBL-Ig) fusion, or afragment thereof that binds 4-1BB). In some embodiments, a vector canencode an agonist of TNFRSF25 (e.g., a TL1A-Ig fusion, or a fragmentthereof that binds TNFRSF25), or an agonist of glucocorticoid-inducedtumor necrosis factor receptor (GITR) (e.g., a GITR ligand-Ig (GITRL-Ig)fusion, or a fragment thereof that binds GITR), or an agonist of CD40(e.g., a CD40 ligand-Ig (CD40L-Ig) fusion, or a fragment thereof thatbinds CD40); or an agonist of CD27 (e.g., a CD27 ligand-Ig (e.g.CD7OL-Ig) fusion, or a fragment thereof that binds CD40).

ICOS is an inducible T cell costimulatory receptor molecule thatdisplays some homology to CD28 and CTLA-4, and interacts with B7-H2expressed on the surface of antigen-presenting cells. ICOS has beenimplicated in the regulation of cell-mediated and humoral immuneresponses.

4-1BB is a type 2 transmembrane glycoprotein belonging to the TNFsuperfamily, and is expressed on activated T Lymphocytes.

OX40 (also referred to as CD134 or TNFRSF4) is a T cell costimulatorymolecule that is engaged by OX40L, and frequently is induced in antigenpresenting cells and other cell types. OX40 is known to enhance cytokineexpression and survival of effector T cells.

GITR (TNFRSF18) is a T cell costimulatory molecule that is engaged byGITRL and is preferentially expressed in FoxP3+ regulatory T cells. GITRplays a significant role in the maintenance and function of Treg withinthe tumor microenvironment.

TNFRSF25 is a T cell costimulatory molecule that is preferentiallyexpressed in CD4+ and CD8+ T cells following antigen stimulation.Signaling through TNFRSF25 is provided by TL1A, and functions to enhanceT cell sensitivity to IL-2 receptor mediated proliferation in a cognateantigen dependent manner.

CD40 is a costimulatory protein found on various antigen presentingcells which plays a role in their activation. The binding of CD40L(CD154) on T_(H) cells to CD40 activates antigen presenting cells andinduces a variety of downstream effects.

CD27 a T cell costimulatory molecule belonging to the TNF superfamilywhich plays a role in the generation and long-term maintenance of T cellimmunity. It binds to a ligand CD70 in various immunological processes.

Additional costimulatory molecules that may be utilized in the presentinvention include, but are not limited to, HVEM, CD28, CD30, CD30L,CD40, CD70, LIGHT (CD258), B7-1, and B7-2.

As for the 96-Ig fusions, the Ig portion (“tag”) of the T cellcostimulatory fusion protein can contain, a non-variable portion of animmunoglobulin molecule (e.g., an IgG1, IgG2, IgG3, IgG4, IgM, IgA, orIgE molecule). As described above, such portions typically contain atleast functional CH2 and CH3 domains of the constant region of animmunoglobulin heavy chain. In some embodiments, a T cell costimulatorypeptide can be fused to the hinge, CH2 and CH3 domains of murine IgG1(Bowen et al., J Immunol 1996, 156:442-449). The Ig tag can be from amammalian (e.g., human, mouse, monkey, or rat) immunoglobulin, but humanimmunoglobulin can be particularly useful when the fusion protein isintended for in vivo use for humans Again, DNAs encoding immunoglobulinlight or heavy chain constant regions are known or readily availablefrom cDNA libraries. Various leader sequences as described above alsocan be used for secretion of T cell costimulatory fusion proteins frombacterial and mammalian cells.

A representative nucleotide optimized sequence (SEQ ID NO:4) encodingthe extracellular domain of human ICOSL fused to Ig, and the amino acidsequence of the encoded fusion (SEQ ID NO:5) are provided:

(SEQ ID NO: 4) ATGAGACTGGGAAGCCCTGGCCTGCTGTTTCTGCTGTTCAGCAGCCTGAGAGCCGACACCCAGGAAAAAGAAGTGCGGGCCATGGTGGGAAGCGACGTGGAACTGAGCTGCGCCTGTCCTGAGGGCAGCAGATTCGACCTGAACGACGTGTACGTGTACTGGCAGACCAGCGAGAGCAAGACCGTCGTGACCTACCACATCCCCCAGAACAGCTCCCTGGAAAACGTGGACAGCCGGTACAGAAACCGGGCCCTGATGTCTCCTGCCGGCATGCTGAGAGGCGACTTCAGCCTGCGGCTGTTCAACGTGACCCCCCAGGACGAGCAGAAATTCCACTGCCTGGTGCTGAGCCAGAGCCTGGGCTTCCAGGAAGTGCTGAGCGTGGAAGTGACCCTGCACGTGGCCGCCAATTTCAGCGTGCCAGTGGTGTCTGCCCCCCACAGCCCTTCTCAGGATGAGCTGACCTTCACCTGTACCAGCATCAACGGCTACCCCAGACCCAATGTGTACTGGATCAACAAGACCGACAACAGCCTGCTGGACCAGGCCCTGCAGAACGATACCGTGTTCCTGAACATGCGGGGCCTGTACGACGTGGTGTCCGTGCTGAGAATCGCCAGAACCCCCAGCGTGAACATCGGCTGCTGCATCGAGAACGTGCTGCTGCAGCAGAACCTGACCGTGGGCAGCCAGACCGGCAACGACATCGGCGAGAGAGACAAGATCACCGAGAACCCCGTGTCCACCGGCGAGAAGAATGCCGCCACCTCTAAGTACGGCCCTCCCTGCCCTTCTTGCCCAGCCCCTGAATTTCTGGGCGGACCCTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGGGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGCAACGCCACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCAGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACTCCCGGCTGACAGTGGACAAGAGCAGCTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAAGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCTGGGCA AATGA (SEQ ID NO: 5)MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

A representative nucleotide optimized sequence (SEQ ID NO:6) encodingthe extracellular domain of human 4-1BBL fused to Ig, and the encodedamino acid sequence (SEQ ID NO:7) are provided:

(SEQ ID NO: 6) ATGTCTAAGTACGGCCCTCCCTGCCCTAGCTGCCCTGCCCCTGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAACGCCACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGCTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGGGCAAGGCCTGTCCATGGGCTGTGTCTGGCGCTAGAGCCTCTCCTGGATCTGCCGCCAGCCCCAGACTGAGAGAGGGACCTGAGCTGAGCCCCGATGATCCTGCCGGACTGCTGGATCTGAGACAGGGCATGTTCGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCCGGCTCTGTGTCTCTGGCTCTGCATCTGCAGCCCCTGAGATCTGCTGCTGGCGCTGCTGCTCTGGCCCTGACAGTGGACCTGCCTCCTGCCTCTAGCGAGGCCAGAAACAGCGCATTCGGGTTTCAAGGCAGACTGCTGCACCTGTCTGCCGGCCAGAGACTGGGAGTGCATCTGCACACAGAGGCCAGAGCCAGGCACGCCTGGCAGCTGACTCAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTAGCCCCAGATCCG AATGA (SEQ ID NO: 7)MSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

A representative nucleotide optimized sequence (SEQ ID NO:8) encodingthe extracellular domain of human TL1A fused to Ig, and the encodedamino acid sequence (SEQ ID NO:9) are provided:

(SEQ ID NO: 8) ATGTCTAAGTACGGCCCTCCCTGCCCTAGCTGCCCTGCCCCTGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAACGCCACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGCTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGGGCAAGATCGAGGGCCGGATGGATAGAGCCCAGGGCGAAGCCTGCGTGCAGTTCCAGGCTCTGAAGGGCCAGGAATTCGCCCCCAGCCACCAGCAGGTGTACGCCCCTCTGAGAGCCGACGGCGATAAGCCTAGAGCCCACCTGACAGTCGTGCGGCAGACCCCTACCCAGCACTTCAAGAATCAGTTCCCCGCCCTGCACTGGGAGCACGAACTGGGCCTGGCCTTCACCAAGAACAGAATGAACTACACCAACAAGTTTCTGCTGATCCCCGAGAGCGGCGACTACTTCATCTACAGCCAAGTGACCTTCCGGGGCATGACCAGCGAGTGCAGCGAGATCAGACAGGCCGGCAGACCTAACAAGCCCGACAGCATCACCGTCGTGATCACCAAAGTGACCGACAGCTACCCCGAGCCCACCCAGCTGCTGATGGGCACCAAGAGCGTGTGCGAAGTGGGCAGCAACTGGTTCCAGCCCATCTACCTGGGCGCCATGTTTAGTCTGCAAGAGGGCGACAAGCTGATGGTCAACGTGTCCGACATCAGCCTGGTGGATTACACCAAAGAGGACAAGACCTTCTTCGGCGCCTTTCTGCTCTGA (SEQ ID NO: 9)MSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIEGRMDRAQGEACVQFQALKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL.

A representative nucleotide optimized sequence (SEQ ID NO:10) encodinghuman OX40L-Ig, and the encoded amino acid sequence (SEQ ID NO:11) areprovided:

(SEQ ID NO: 10) ATGTCTAAGTACGGCCCTCCCTGCCCTAGCTGCCCTGCCCCTGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAACGCCACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGCTGGCAGGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGGGCAAGATCGAGGGCCGGATGGATCAGGTGTCACACAGATACCCCCGGATCCAGAGCATCAAAGTGCAGTTTACCGAGTACAAGAAAGAGAAGGGCTTTATCCTGACCAGCCAGAAAGAGGACGAGATCATGAAGGTGCAGAACAACAGCGTGATCATCAACTGCGACGGGTTCTACCTGATCAGCCTGAAGGGCTACTTCAGTCAGGAAGTGAACATCAGCCTGCACTACCAGAAGGACGAGGAACCCCTGTTCCAGCTGAAGAAAGTGCGGAGCGTGAACAGCCTGATGGTGGCCTCTCTGACCTACAAGGACAAGGTGTACCTGAACGTGACCACCGACAACACCAGCCTGGACGACTTCCACGTGAACGGCGGCGAGCTGATCCTGATTCACCAGAACCCCGGCGAGTTCTGCGT GCTCTGA (SEQ ID NO:11) MSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKIEGRMDQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHV NGGELILIHQNPGEFCVL.

Representative nucleotide and amino acid sequences for human TL1A areset forth in SEQ ID NO:12 and SEQ ID NO:13, respectively:

(SEQ ID NO: 12) TCCCAAGTAGCTGGGACTACAGGAGCCCACCACCACCCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAAGATGGTCTTGATCACCTGACCTCGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGCGCCCGGCCTCCATTCAAGTCTTTATTGAATATCTGCTATGTTCTACACACTGTTCTAGGTGCTGGGGATGCAACAGGGGACAAAATAGGCAAAATCCCTGTCCTTTTGGGGTTGACATTCTAGTGACTCTTCATGTAGTCTAGAAGAAGCTCAGTGAATAGTGTCTGTGGTTGTTACCAGGGACACAATGACAGGAACATTCTTGGGTAGAGTGAGAGGCCTGGGGAGGGAAGGGTCTCTAGGATGGAGCAGATGCTGGGCAGTCTTAGGGAGCCCCTCCTGGCATGCACCCCCTCATCCCTCAGGCCACCCCCGTCCCTTGCAGGAGCACCCTGGGGAGCTGTCCAGAGCGCTGTGCCGCTGTCTGTGGCTGGAGGCAGAGTAGGTGGTGTGCTGGGAATGCGAGTGGGAGAACTGGGATGGACCGAGGGGAGGCGGGTGAGGAGGGGGGCAACCACCCAACACCCACCAGCTGCTTTCAGTGTTCTGGGTCCAGGTGCTCCTGGCTGGCCTTGTGGTCCCCCTCCTGCTTGGGGCCACCCTGACCTACACATACCGCCACTGCTGGCCTCACAAGCCCCTGGTTACTGCAGATGAAGCTGGGATGGAGGCTCTGACCCCACCACCGGCCACCCATCTGTCACCCTTGGACAGCGCCCACACCCTTCTAGCACCTCCTGACAGCAGTGAGAAGATCTGCACCGTCCAGTTGGTGGGTAACAGCTGGACCCCTGGCTACCCCGAGACCCAGGAGGCGCTCTGCCCGCAGGTGACATGGTCCTGGGACCAGTTGCCCAGCAGAGCTCTTGGCCCCGCTGCTGCGCCCACACTCTCGCCAGAGTCCCCAGCCGGCTCGCCAGCCATGATGCTGCAGCCGGGCCCGCAGCTCTACGACGTGATGGACGCGGTCCCAGCGCGGCGCTGGAAGGAGTTCGTGCGCACGCTGGGGCTGCGCGAGGCAGAGATCGAAGCCGTGGAGGTGGAGATCGGCCGCTTCCGAGACCAGCAGTACGAGATGCTCAAGCGCTGGCGCCAGCAGCAGCCCGCGGGCCTCGGAGCCGTTTACGCGGCCCTGGAGCGCATGGGGCTGGACGGCTGCGTGGAAGACTTGCGCAGCCGCCTGCAGCGCGGCCCGTGACACGGCGCCCACTTGCCACCTAGGCGCTCTGGTGGCCCTTGCAGAAGCCCTAAGTACGGTTACTTATGCGTGTAGACATTTTATGTCACTTATTAAGCCGCTGGCACGGCCCTGCGTAGCAGCACCAGCCGGCCCCACCCCTGCTCGCCCCTATCGCTCCAGCCAAGGCGAAGAAGCACGAACGAATGTCGAGAGGGGGTGAAGACATTTCTCAACTTCTCGGCCGGAGTTTGGCTGAGATCGCGGTATTAAATCTGTGAAAGAAAACAAAACAAAACAA (SEQ ID NO: 13)MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCGNSTCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVADTRCGCKPGWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVSCPTPPPSLAGAPWGAVQSAVPLSVAGGRVGVFWVQVLLAGLVVPLLLGATLTYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP.

Representative nucleotide and amino acid sequences for human HVEM areset forth in SEQ ID NO:38 (accession no. CR456909) and SEQ ID NO:39,respectively (accession no. CR456909):

(SEQ ID NO: 38) ATGGAGCCTCCTGGAGACTGGGGGCCTCCTCCCTGGAGATCCACCCCCAAAACCGACGTCTTGAGGCTGGTGCTGTATCTCACCTTCCTGGGAGCCCCCTGCTACGCCCCAGCTCTGCCGTCCTGCAAGGAGGACGAGTACCCAGTGGGCTCCGAGTGCTGCCCCAAGTGCAGTCCAGGTTATCGTGTGAAGGAGGCCTGCGGGGAGCTGACGGGCACAGTGTGTGAACCCTGCCCTCCAGGCACCTACATTGCCCACCTCAATGGCCTAAGCAAGTGTCTGCAGTGCCAAATGTGTGACCCAGCCATGGGCCTGCGCGCGAGCCGGAACTGCTCCAGGACAGAGAACGCCGTGTGTGGCTGCAGCCCAGGCCACTTCTGCATCGTCCAGGACGGGGACCACTGCGCCGCGTGCCGCGCTTACGCCACCTCCAGCCCGGGCCAGAGGGTGCAGAAGGGAGGCACCGAGAGTCAGGACACCCTGTGTCAGAACTGCCCCCCGGGGACCTTCTCTCCCAATGGGACCCTGGAGGAATGTCAGCACCAGACCAAGTGCAGCTGGCTGGTGACGAAGGCCGGAGCTGGGACCAGCAGCTCCCACTGGGTATGGTGGTTTCTCTCAGGGAGCCTCGTCATCGTCATTGTTTGCTCCACAGTTGGCCTAATCATATGTGTGAAAAGAAGAAAGCCAAGGGGTGATGTAGTCAAGGTGATCGTCTCCGTCCAGCGGAAAAGACAGGAGGCAGAAGGTGAGGCCACAGTCATTGAGGCCCTGCAGGCCCCTCCGGACGTCACCACGGTGGCCGTGGAGGAGACAATACCCTCATTCACGGGGAGGAGCCCAAACCATT AA (SEQ ID NO: 39)MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH.

Representative nucleotide and amino acid sequences for human CD28 areset forth in SEQ ID NO:40 (accession no. NM_006139) and SEQ ID NO:41,respectively:

(SEQ ID NO: 40) TAAAGTCATCAAAACAACGTTATATCCTGTGTGAAATGCTGCAGTCAGGATGCCTTGTGGTTTGAGTGCCTTGATCATGTGCCCTAAGGGGATGGTGGCGGTGGTGGTGGCCGTGGATGACGGAGACTCTCAGGCCTTGGCAGGTGCGTCTTTCAGTTCCCCTCACACTTCGGGTTCCTCGGGGAGGAGGGGCTGGAACCCTAGCCCATCGTCAGGACAAAGATGCTCAGGCTGCTCTTGGCTCTCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATTTTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATGCGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCAAGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTGCTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAGCTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGAAATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTGTATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCTGACACGGACGCCTATCCAGAAGCCAGCCGGCTGGCAGCCCCCATCTGCTCAATATCACTGCTCTGGATAGGAAATGACCGCCATCTCCAGCCGGCCACCTCAGGCCCCTGTTGGGCCACCAATGCCAATTTTTCTCGAGTGACTAGACCAAATATCAAGATCATTTTGAGACTCTGAAATGAAGTAAAAGAGATTTCCTGTGACAGGCCAAGTCTTACAGTGCCATGGCCCACATTCCAACTTACCATGTACTTAGTGACTTGACTGAGAAGTTAGGGTAGAAAACAAAAAGGGAGTGGATTCTGGGAGCCTCTTCCCTTTCTCACTCACCTGCACATCTCAGTCAAGCAAAGTGTGGTATCCACAGACATTTTAGTTGCAGAAGAAAGGCTAGGAAATCATTCCTTTTGGTTAAATGGGTGTTTAATCTTTTGGTTAGTGGGTTAAACGGGGTAAGTTAGAGTAGGGGGAGGGATAGGAAGACATATTTAAAAACCATTAAAACACTGTCTCCCACTCATGAAATGAGCCACGTAGTTCCTATTTAATGCTGTTTTCCTTTAGTTTAGAAATACATAGACATTGTCTTTTATGAATTCTGATCATATTTAGTCATTTTGACCAAATGAGGGATTTGGTCAAATGAGGGATTCCCTCAAAGCAATATCAGGTAAACCAAGTTGCTTTCCTCACTCCCTGTCATGAGACTTCAGTGTTAATGTTCACAATATACTTTCGAAAGAATAAAATAGTTCTCCTACATGAAGAAAGAATATGTCAGGAAATAAGGTCACTTTATGTCAAAATTATTTGAGTACTATGGGACCTGGCGCAGTGGCTCATGCTTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACTTGAGATCAGGACCAGCCTGGTCAAGATGGTGAAACTCCGTCTGTACTAAAAATACAAAATTTAGCTTGGCCTGGTGGCAGGCACCTGTAATCCCAGCTGCCCAAGAGGCTGAGGCATGAGAATCGCTTGAACCTGGCAGGCGGAGGTTGCAGTGAGCCGAGATAGTGCCACAGCTCTCCAGCCTGGGCGACAGAGTGAGACTCCATCTCAAACAACAACAACAACAACAACAACAACAACAAACCACAAAATTATTTGAGTACTGTGAAGGATTATTTGTCTAACAGTTCATTCCAATCAGACCAGGTAGGAGCTTTCCTGTTTCATATGTTTCAGGGTTGCACAGTTGGTCTCTTTAATGTCGGTGTGGAGATCCAAAGTGGGTTGTGGAAAGAGCGTCCATAGGAGAAGTGAGAATACTGTGAAAAAGGGATGTTAGCATTCATTAGAGTATGAGGATGAGTCCCAAGAAGGTTCTTTGGAAGGAGGACGAATAGAATGGAGTAATGAAATTCTTGCCATGTGCTGAGGAGATAGCCAGCATTAGGTGACAATCTTCCAGAAGTGGTCAGGCAGAAGGTGCCCTGGTGAGAGCTCCTTTACAGGGACTTTATGTGGTTTAGGGCTCAGAGCTCCAAAACTCTGGGCTCAGCTGCTCCTGTACCTTGGAGGTCCATTCACATGGGAAAGTATTTTGGAATGTGTCTTTTGAAGAGAGCATCAGAGTTCTTAAGGGACTGGGTAAGGCCTGACCCTGAAATGACCATGGATATTTTTCTACCTACAGTTTGAGTCAACTAGAATATGCCTGGGGACCTTGAAGAATGGCCCTTCAGTGGCCCTCACCATTTGTTCATGCTTCAGTTAATTCAGGTGTTGAAGGAGCTTAGGTTTTAGAGGCACGTAGACTTGGTTCAAGTCTCGTTAGTAGTTGAATAGCCTCAGGCAAGTCACTGCCCACCTAAGATGATGGTTCTTCAACTATAAAATGGAGATAATGGTTACAAATGTCTCTTCCTATAGTATAATCTCCATAAGGGCATGGCCCAAGTCTGTCTTTGACTCTGCCTATCCCTGACATTTAGTAGCATGCCCGACATACAATGTTAGCTATTGGTATTATTGCCATATAGATAAATTATGTATAAAAATTAAACTGGGCAATAGCCTAAGAAGGGGGGAATATTGTAACACAAATTTAAACCCACTACGCAGGGATGAGGTGCTATAATATGAGGACCTTTTAACTTCCATCATTTTCCTGTTTCTTGAAATAGTTTATCTTGTAATGAAATATAAGGCACCTCCCACTTTTATGTATAGAAAGAGGTCTTTTAATTTTTTTTTAATGTGAGAAGGAAGGGAGGAGTAGGAATCTTGAGATTCCAGATCGAAAATACTGTACTTTGGTTGATTTTTAAGTGGGCTTCCATTCCATGGATTTAATCAGTCCCAAGAAGATCAAACTCAGCAGTACTTGGGTGCTGAAGAACTGTTGGATTTACCCTGGCACGTGTGCCACTTGCCAGCTTCTTGGGCACACAGAGTTCTTCAATCCAAGTTATCAGATTGTATTTGAAAATGACAGAGCTGGAGAGTTTTTTGAAATGGCAGTGGCAAATAAATAAATACTTTTTTTTAAATGGAAAGACTTGATCTATGGTAATAAATGATTTTGTTTTCTGACTGGAAAAATAGGCCTACTAAAGATGAATCACACTTGAGATGTTTCTTACTCACTCTGCACAGAAACAAAGAAGAAATGTTATACAGGGAAGTCCGTTTTCACTATTAGTATGAACCAAGAAATGGTTCAAAAACAGTGGTAGGAGCAATGCTTTCATAGTTTCAGATATGGTAGTTATGAAGAAAACAATGTCATTTGCTGCTATTATTGTAAGAGTCTTATAATTAATGGTACTCCTATAATTTTTGATTGTGAGCTCACCTATTTGGGTTAAGCATGCCAATTTAAAGAGACCAAGTGTATGTACATTATGTTCTACATATTCAGTGATAAAATTACTAAACTACTATATGTCTGCTTTAAATTTGTACTTTAATATTGTCTTTTGGTATTAAGAAAGATATGCTTTCAGAATAGATATGCTTCGCTTTGGCAAGGAATTTGGATAGAACTTGCTATTTAAAAGAGGTGTGGGGTAAATCCTTGTATAAATCTCCAGTTTAGCCTTTTTTGAAAAAGCTAGACTTTCAAATACTAATTTCACTTCAAGCAGGGTACGTTTCTGGTTTGTTTGCTTGACTTCAGTCACAATTTCTTATCAGACCAATGGCTGACCTCTTTGAGATGTCAGGCTAGGCTTACCTATGTGTTCTGTGTCATGTGAATGCTGAGAAGTTTGACAGAGATCCAACTTCAGCCTTGACCCCATCAGTCCCTCGGGTTAACTAACTGAGCCACCGGTCCTCATGGCTATTTTAATGAGGGTATTGATGGTTAAATGCATGTCTGATCCCTTATCCCAGCCATTTGCACTGCCAGCTGGGAACTATACCAGACCTGGATACTGATCCCAAAGTGTTAAATTCAACTACATGCTGGAGATTAGAGATGGTGCCAATAAAGGACCCAGAACCAGGATCTTGATTGCTATAGACTTATTAATAATCCAGGTCAAAGAGAGTGACACACACTCTCTCAAGACCTGGGGTGAGGGAGTCTGTGTTATCTGCAAGGCCATTTGAGGCTCAGAAAGTCTCTCTTTCCTATAGATATATGCATACTTTCTGACATATAGGAATGTATCAGGAATACTCAACCATCACAGGCATGTTCCTACCTCAGGGCCTTTACATGTCCTGTTTACTCTGTCTAGAATGTCCTTCTGTAGATGACCTGGCTTGCCTCGTCACCCTTCAGGTCCTTGCTCAAGTGTCATCTTCTCCCCTAGTTAAACTACCCCACACCCTGTCTGCTTTCCTTGCTTATTTTTCTCCATAGCATTTTACCATCTCTTACATTAGACATTTTTCTTATTTATTTGTAGTTTATAAGCTTCATGAGGCAAGTAACTTTGCTTTGTTTCTTGCTGTATCTCCAGTGCCCAGAGCAGTGCCTGGTATATAATAAATATTTATTGACTGAGTGAAAAAAAAAAAAAAAAA (SEQ ID NO: 41)MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRS.

Representative nucleotide and amino acid sequences for human CD30L areset forth in SEQ ID NO:42 (accession no. L09753) and SEQ ID NO:43,respectively:

(SEQ ID NO: 42) CCAAGTCACATGATTCAGGATTCAGGGGGAGAATCCTTCTTGGAACAGAGATGGGCCCAGAACTGAATCAGATGAAGAGAGATAAGGTGTGATGTGGGGAAGACTATATAAAGAATGGACCCAGGGCTGCAGCAAGCACTCAACGGAATGGCCCCTCCTGGAGACACAGCCATGCATGTGCCGGCGGGCTCCGTGGCCAGCCACCTGGGGACCACGAGCCGCAGCTATTTCTATTTGACCACAGCCACTCTGGCTCTGTGCCTTGTCTTCACGGTGGCCACTATTATGGTGTTGGTCGTTCAGAGGACGGACTCCATTCCCAACTCACCTGACAACGTCCCCCTCAAAGGAGGAAATTGCTCAGAAGACCTCTTATGTATCCTGAAAAGAGCTCCATTCAAGAAGTCATGGGCCTACCTCCAAGTGGCAAAGCATCTAAACAAAACCAAGTTGTCTTGGAACAAAGATGGCATTCTCCATGGAGTCAGATATCAGGATGGGAATCTGGTGATCCAATTCCCTGGTTTGTACTTCATCATTTGCCAACTGCAGTTTCTTGTACAATGCCCAAATAATTCTGTCGATCTGAAGTTGGAGCTTCTCATCAACAAGCATATCAAAAAACAGGCCCTGGTGACAGTGTGTGAGTCTGGAATGCAAACGAAACACGTATACCAGAATCTCTCTCAATTCTTGCTGGATTACCTGCAGGTCAACACCACCATATCAGTCAATGTGGATACATTCCAGTACATAGATACAAGCACCTTTCCTCTTGAGAATGTGTTGTCCATCTTCTTATACAGTAATTCAGACTGAACAGTTTCTCTTGGCCTTCAGGAAGAAAGCGCCTCTCTACCATACAGTATTTCATCCCTCCAAACACTTGGGCAAAAAGAAAACTTTAGACCAAGACAAACTACACAGGGTATTAAATAGTATACTTCTCCTTCTGTCTCTTGGAAAGATACAGCTCCAGGGTTAAAAAGAGAGTTTTTAGTGAAGTATCTTTCAGATAGCAGGCAGGGAAGCAATGTAGTGTGGTGGGCAGAGCCCCACACAGAATCAGAAGGGATGAATGGATGTCCCAGCCCAACCACTAATTCACTGTATGGTCTTGATCTATTTCTTCTGTTTTGAGAGCCTCCAGTTAAAATGGGGCTTCAGTACCAGAGCAGCTAGCAACTCTGCCCTAATGGGAAATGAAGGGGAGCTGGGTGTGAGTGTTTACACTGTGCCCTTCACGGGATACTTCTTTTATCTGCAGATGGCCTAATGCTTAGTTGTCCAAGTCGCGATCAAGGACTCTCTCACACAGGAAACTTCCCTATACTGGCAGATACACTTGTGACTGAACCATGCCCAGTTTATGCCTGTCTGACTGTCACTCTGGCACTAGGAGGCTGATCTTGTACTCCATATGACCCCACCCCTAGGAACCCCCAGGGAAAACCAGGCTCGGACAGCCCCCTGTTCCTGAGATGGAAAGCACAAATTTAATACACCACCACAATGGAAAACAAGTTCAAAGACTTTTACTTACAGATCCTGGACAGAAAGGGCATAATGAGTCTGAAGGGCAGTCCTCCTTCTCCAGGTTACATGAGGCAGGAATAAGAAGTCAGACAGAGACAGCAAGACAGTTAACAACGTAGGTAAAGAAATAGGGTGTGGTCACTCTCAATTCACTGGCAAATGCCTGAATGGTCTGTCTGAAGGAAGCAACAGAGAAGTGGGGAATCCAGTCTGCTAGGCAGGAAAGATGCCTCTAAGTTCTTGTCTCTGGCCAGAGGTGTGGTATAGAACCAGAAACCCATATCAAGGGTGACTAAGCCCGGCTTCCGGTATGAGAAATTAAACTTGTATACAAAATGGTTGCCAAGGCAACATAAAATTATAA GAATTC (SEQ ID NO:43) MDPGLQQALNGMAPPGDTAMHVPAGSVASHLGTTSRSYFYLTTATLALCLVFTVATIMVLVVQRTDSIPNSPDNVPLKGGNCSEDLLCILKRAPFKKSWAYLQVAKHLNKTKLSWNKDGILHGVRYQDGNLVIQFPGLYFIICQLQFLVQCPNNSVDLKLELLINKHIKKQALVTVCESGMQTKHVYQNLSQFLLDYLQVNTTISVNVDTFQYIDTSTFPLENVLSIFLYSNSD.

Representative nucleotide and amino acid sequences for human CD40 areset forth in SEQ ID NO:44 (accession no. NM_001250) and SEQ ID NO:45,respectively:

(SEQ ID NO: 44) TTTCCTGGGCGGGGCCAAGGCTGGGGCAGGGGAGTCAGCAGAGGCCTCGCTCGGGCGCCCAGTGGTCCTGCCGCCTGGTCTCACCTCGCTATGGTTCGTCTGCCTCTGCAGTGCGTCCTCTGGGGCTGCTTGCTGACCGCTGTCCATCCAGAACCACCCACTGCATGCAGAGAAAAACAGTACCTAATAAACAGTCAGTGCTGTTCTTTGTGCCAGCCAGGACAGAAACTGGTGAGTGACTGCACAGAGTTCACTGAAACGGAATGCCTTCCTTGCGGTGAAAGCGAATTCCTAGACACCTGGAACAGAGAGACACACTGCCACCAGCACAAATACTGCGACCCCAACCTAGGGCTTCGGGTCCAGCAGAAGGGCACCTCAGAAACAGACACCATCTGCACCTGTGAAGAAGGCTGGCACTGTACGAGTGAGGCCTGTGAGAGCTGTGTCCTGCACCGCTCATGCTCGCCCGGCTTTGGGGTCAAGCAGATTGCTACAGGGGTTTCTGATACCATCTGCGAGCCCTGCCCAGTCGGCTTCTTCTCCAATGTGTCATCTGCTTTCGAAAAATGTCACCCTTGGACAAGCTGTGAGACCAAAGACCTGGTTGTGCAACAGGCAGGCACAAACAAGACTGATGTTGTCTGTGGTCCCCAGGATCGGCTGAGAGCCCTGGTGGTGATCCCCATCATCTTCGGGATCCTGTTTGCCATCCTCTTGGTGCTGGTCTTTATCAAAAAGGTGGCCAAGAAGCCAACCAATAAGGCCCCCCACCCCAAGCAGGAACCCCAGGAGATCAATTTTCCCGACGATCTTCCTGGCTCCAACACTGCTGCTCCAGTGCAGGAGACTTTACATGGATGCCAACCGGTCACCCAGGAGGATGGCAAAGAGAGTCGCATCTCAGTGCAGGAGAGACAGTGAGGCTGCACCCACCCAGGAGTGTGGCCACGTGGGCAAACAGGCAGTTGGCCAGAGAGCCTGGTGCTGCTGCTGCTGTGGCGTGAGGGTGAGGGGCTGGCACTGACTGGGCATAGCTCCCCGCTTCTGCCTGCACCCCTGCAGTTTGAGACAGGAGACCTGGCACTGGATGCAGAAACAGTTCACCTTGAAGAACCTCTCACTTCACCCTGGAGCCCATCCAGTCTCCCAACTTGTATTAAAGACAGAGGCAGAAGTTTGGTGGTGGTGGTGTTGGGGTATGGTTTAGTAATATCCACCAGACCTTCCGATCCAGCAGTTTGGTGCCCAGAGAGGCATCATGGTGGCTTCCCTGCGCCCAGGAAGCCATATACACAGATGCCCATTGCAGCATTGTTTGTGATAGTGAACAACTGGAAGCTGCTTAACTGTCCATCAGCAGGAGACTGGCTAAATAAAATTAGAATATATTTATACAACAGAATCTCAAAAACACTGTTGAGTAAGGAAAAAAAGGCATGCTGCTGAATGATGGGTATGGAACTTTTTAAAAAAGTACATGCTTTTATGTATGTATATTGCCTATGGATATATGTATAAATACAATATGCATCATATATTGATATAACAAGGGTTCTGGAAGGGTACACAGAAAACCCACAGCTCGAAGAGTGGTGACGTCTGGGGTGGGGAAGAAGGGTCTGGGGG (SEQ ID NO: 45)MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ.

Representative nucleotide and amino acid sequences for human CD70 areset forth in SEQ ID NO:46 (accession no. NM_001252) and SEQ ID NO:47,respectively:

(SEQ ID NO: 46) CCAGAGAGGGGCAGGCTGGTCCCCTGACAGGTTGAAGCAAGTAGACGCCCAGGAGCCCCGGGAGGGGGCTGCAGTTTCCTTCCTTCCTTCTCGGCAGCGCTCCGCGCCCCCATCGCCCCTCCTGCGCTAGCGGAGGTGATCGCCGCGGCGATGCCGGAGGAGGGTTCGGGCTGCTCGGTGCGGCGCAGGCCCTATGGGTGCGTCCTGCGGGCTGCTTTGGTCCCATTGGTCGCGGGCTTGGTGATCTGCCTCGTGGTGTGCATCCAGCGCTTCGCACAGGCTCAGCAGCAGCTGCCGCTCGAGTCACTTGGGTGGGACGTAGCTGAGCTGCAGCTGAATCACACAGGACCTCAGCAGGACCCCAGGCTATACTGGCAGGGGGGCCCAGCACTGGGCCGCTCCTTCCTGCATGGACCAGAGCTGGACAAGGGGCAGCTACGTATCCATCGTGATGGCATCTACATGGTACACATCCAGGTGACGCTGGCCATCTGCTCCTCCACGACGGCCTCCAGGCACCACCCCACCACCCTGGCCGTGGGAATCTGCTCTCCCGCCTCCCGTAGCATCAGCCTGCTGCGTCTCAGCTTCCACCAAGGTTGTACCATTGCCTCCCAGCGCCTGACGCCCCTGGCCCGAGGGGACACACTCTGCACCAACCTCACTGGGACACTTTTGCCTTCCCGAAACACTGATGAGACCTTCTTTGGAGTGCAGTGGGTGCGCCCCTGACCACTGCTGCTGATTAGGGTTTTTTAAATTTTATTTTATTTTATTTAAGTTCAAGAGAAAAAGTGTACACACAGGGGCCACCCGGGGTTGGGGTGGGAGTGTGGTGGGGGGTAGTGGTGGCAGGACAAGAGAAGGCATTGAGCTTTTTCTTTCATTTTCCTATTAAAA AATACAAAAATCA (SEQ IDNO: 47) MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP.

Representative nucleotide and amino acid sequences for human LIGHT areset forth in SEQ ID NO:48 (accession no. CR541854) and SEQ ID NO:49,respectively:

(SEQ ID NO: 48) ATGGAGGAGAGTGTCGTACGGCCCTCAGTGTTTGTGGTGGATGGACAGACCGACATCCCATTCACGAGGCTGGGACGAAGCCACCGGAGACAGTCGTGCAGTGTGGCCCGGGTGGGTCTGGGTCTCTTGCTGTTGCTGATGGGGGCCGGGCTGGCCGTCCAAGGCTGGTTCCTCCTGCAGCTGCACTGGCGTCTAGGAGAGATGGTCACCCGCCTGCCTGACGGACCTGCAGGCTCCTGGGAGCAGCTGATACAAGAGCGAAGGTCTCACGAGGTCAACCCAGCAGCGCATCTCACAGGGGCCAACTCCAGCTTGACCGGCAGCGGGGGGCCGCTGTTATGGGAGACTCAGCTGGGCCTGGCCTTCCTGAGGGGCCTCAGCTACCACGATGGGGCCCTTGTGGTCACCAAAGCTGGCTACTACTACATCTACTCCAAGGTGCAGCTGGGCGGTGTGGGCTGCCCGCTGGGCCTGGCCAGCACCATCACCCACGGCCTCTACAAGCGCACACCCCGCTACCCCGAGGAGCTGGAGCTGTTGGTCAGCCAGCAGTCACCCTGCGGACGGGCCACCAGCAGCTCCCGGGTCTGGTGGGACAGCAGCTTCCTGGGTGGTGTGGTACACCTGGAGGCTGGGGAGGAGGTGGTCGTCCGTGTGCTGGATGAACGCCTGGTTCGACTGCGTGATGGTACCCGGTCTTACTTCGGGGCTTTCATGGTGTGA (SEQ ID NO: 49)MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV.

In various embodiments, the present invention provides for variantscomprising any of the sequences described herein, for instance, asequence having at least about 60%, or at least about 61%, or at leastabout 62%, or at least about 63%, or at least about 64%, or at leastabout 65%, or at least about 66%, or at least about 67%, or at leastabout 68%, or at least about 69%, or at least about 70%, or at leastabout 71%, or at least about 72%, or at least about 73%, or at leastabout 74%, or at least about 75%, or at least about 76%, or at leastabout 77%, or at least about 78%, or at least about 79%, or at leastabout 80%, or at least about 81%, or at least about 82%, or at leastabout 83%, or at least about 84%, or at least about 85%, or at leastabout 86%, or at least about 87%, or at least about 88%, or at leastabout 89%, or at least about 90%, or at least about 91%, or at leastabout 92%, or at least about 93%, or at least about 94%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99%) sequence identity with any of thesequences disclosed herein (for example, SEQ ID NOS: 1-13 and 38-49).

In various embodiments, the present invention provides for an amino acidsequence having one or more amino acid mutations relative any of theprotein sequences described herein. In some embodiments, the one or moreamino acid mutations may be independently selected from conservative ornon-conservative substitutions, insertions, deletions, and truncationsas described herein.

Checkpoint Blockade/Blockage of Tumor Immunosuppression

Some human tumors can be eliminated by a patient's immune system. Forexample, administration of a monoclonal antibody targeted to an immune“checkpoint” molecule can lead to complete response and tumor remission.A mode of action of such antibodies is through inhibition of an immuneregulatory molecule that the tumors have co-opted as protection from ananti-tumor immune response. By inhibiting these “checkpoint” molecules(e.g., with an antagonistic antibody), a patient's CD8+ T cells may beallowed to proliferate and destroy tumor cells.

For example, administration of a monoclonal antibody targeted to by wayof example, without limitation, CTLA-4 or PD-1 can lead to completeresponse and tumor remission. The mode of action of such antibodies isthrough inhibition of CTLA-4 or PD-1 that the tumors have co-opted asprotection from an anti-tumor immune response. By inhibiting these“checkpoint” molecules (e.g., with an antagonistic antibody), apatient's CD8+ T cells may be allowed to proliferate and destroy tumorcells.

Thus, the vectors provided herein can be used in combination with one ormore blocking antibodies targeted to an immune “checkpoint” molecule.For instance, in some embodiments, the vectors provided herein can beused in combination with one or more blocking antibodies targeted to amolecule such as CTLA-4 or PD-1. For example, the vectors providedherein may be used in combination with an agent that blocks, reducesand/or inhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 withPD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011, CURE TECH),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE)).In an embodiment, the vectors provided herein may be used in combinationwith an agent that blocks, reduces and/or inhibits the activity ofCTLA-4 and/or the binding of CTLA-4 with one or more receptors (e.g.CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For instance, in someembodiments, the immune-modulating agent is an antibody such as, by wayof non-limitation, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and/ortremelimumab (Pfizer). Blocking antibodies against these molecules canbe obtained from, for example, Bristol Myers Squibb (New York, N.Y.),Merck (Kenilworth, N.J.), MedImmune (Gaithersburg, Md.), and Pfizer (NewYork, N.Y.).

Further, the vectors provided herein can be used in combination with oneor more blocking antibodies targeted to an immune “checkpoint” moleculesuch as for example, BTLA, HVEM, TIM3, GALS, LAG3, VISTA, KIR, 2B4,CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases,A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), GITR, GITRL,galectin-9, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2 andvarious B-7 family ligands (including, but are not limited to, B7-1,B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7).

Vectors and Host Cells

This document provides nucleic acid constructs that encode a vaccineprotein fusion protein (e.g., a gp96-Ig fusion protein) and a T cellcostimulatory fusion protein that can be expressed in prokaryotic andeukaryotic cells. For example, this document provides expression vectors(e.g., DNA- or RNA-based vectors) containing nucleotide sequences thatencode a vaccine protein fusion (e.g., a gp96-Ig fusion) and a T cellcostimulatory fusion protein (e.g., OX40L-Ig or a portion thereof thatbinds specifically to OX40, ICOSL-Ig or a portion thereof that bindsspecifically to ICOS, 4-1BBL-Ig, or a portion thereof that bindsspecifically to 4-1BBR, CD40L-Ig, or a portion thereof that bindsspecifically to CD40, CD70-Ig, or a portion thereof that bindsspecifically to CD27, TL1A-Ig or a portion thereof that bindsspecifically to TNFRSF25, or GITRL-Ig or a portion thereof that bindsspecifically to GITR). In addition, this document provides methods formaking the vectors described herein, as well as methods for introducingthe vectors into appropriate host cells for expression of the encodedpolypeptides. In general, the methods provided herein includeconstructing nucleic acid sequences encoding a vaccine protein fusionprotein (e.g., a gp96-Ig fusion protein) and a T cell costimulatoryfusion protein, cloning the sequences encoding the fusion proteins intoan expression vector. The expression vector can be introduced into hostcells or incorporated into virus particles, either of which can beadministered to a subject to, for example, treat cancer or infection.For example, gp96-Ig based vaccines can be generated to stimulateantigen specific immune responses against individual antigens expressedby simian immunodeficiency virus, human immunodeficiency virus,hepatitis C virus and malaria Immune responses to these vaccines may beenhanced through co-expression of a T cell costimulatory fusion proteinby the gp96-Ig vector.

cDNA or DNA sequences encoding a vaccine protein fusion (e.g., a gp96-Igfusion) and a T cell costimulatory fusion protein can be obtained (and,if desired, modified) using conventional DNA cloning and mutagenesismethods, DNA amplification methods, and/or synthetic methods. Ingeneral, a sequence encoding a vaccine protein fusion protein (e.g., agp96-Ig fusion protein) and/or a T cell costimulatory fusion protein canbe inserted into a cloning vector for genetic modification andreplication purposes prior to expression. Each coding sequence can beoperably linked to a regulatory element, such as a promoter, forpurposes of expressing the encoded protein in suitable host cells invitro and in vivo.

Expression vectors can be introduced into host cells for producingsecreted vaccine proteins (e.g., gp96-Ig) and T cell costimulatoryfusion proteins. There are a variety of techniques available forintroducing nucleic acids into viable cells. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,polymer-based systems, DEAE-dextran, viral transduction, the calciumphosphate precipitation method, etc. For in vivo gene transfer, a numberof techniques and reagents may also be used, including liposomes;natural polymer-based delivery vehicles, such as chitosan and gelatin;viral vectors are also suitable for in vivo transduction. In somesituations it is desirable to provide a targeting agent, such as anantibody or ligand specific for a cell surface membrane protein. Whereliposomes are employed, proteins which bind to a cell surface membraneprotein associated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990).

Where appropriate, gene delivery agents such as, e.g., integrationsequences can also be employed. Numerous integration sequences are knownin the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406,1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra etal., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These includerecombinases and transposases. Examples include Cre (Sternberg andHamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247,543-545, 1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R(Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see,e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as AAV, retroviruses, andantiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofAAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).

Cells may be cultured in vitro or genetically engineered, for exampleHost cells can be obtained from normal or affected subjects, includinghealthy humans, cancer patients, and patients with an infectiousdisease, private laboratory deposits, public culture collections such asthe American Type Culture Collection, or from commercial suppliers.

Cells that can be used for production and secretion of gp96-Ig fusionproteins and T cell costimulatory fusion proteins in vivo include,without limitation, epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, or granulocytes, various stem or progenitorcells, such as hematopoietic stem or progenitor cells (e.g., as obtainedfrom bone marrow), umbilical cord blood, peripheral blood, fetal liver,etc., and tumor cells (e.g., human tumor cells). The choice of cell typedepends on the type of tumor or infectious disease being treated orprevented, and can be determined by one of skill in the art.

Different host cells have characteristic and specific mechanisms forpost-translational processing and modification of proteins. A host cellmay be chosen which modifies and processes the expressed gene productsin a specific fashion similar to the way the recipient processes itsheat shock proteins (hsps). For the purpose of producing large amountsof gp96-Ig, it can be preferable that the type of host cell has beenused for expression of heterologous genes, and is reasonably wellcharacterized and developed for large-scale production processes. Insome embodiments, the host cells are autologous to the patient to whomthe present fusion or recombinant cells secreting the present fusionproteins are subsequently administered.

In some embodiments, an expression construct as provided herein can beintroduced into an antigenic cell. As used herein, antigenic cells caninclude preneoplastic cells that are infected with a cancer-causinginfectious agent, such as a virus, but that are not yet neoplastic, orantigenic cells that have been exposed to a mutagen or cancer-causingagent, such as a DNA-damaging agent or radiation, for example. Othercells that can be used are preneoplastic cells that are in transitionfrom a normal to a neoplastic form as characterized by morphology orphysiological or biochemical function.

Typically, the cancer cells and preneoplastic cells used in the methodsprovided herein are of mammalian origin. Mammals contemplated includehumans, companion animals (e.g., dogs and cats), livestock animals(e.g., sheep, cattle, goats, pigs and horses), laboratory animals (e.g.,mice, rats and rabbits), and captive or free wild animals.

In some embodiments, cancer cells (e.g., human tumor cells) can be usedin the methods described herein. The cancer cells provide antigenicpeptides that become associated non-covalently with the expressedgp96-Ig fusion proteins. Cell lines derived from a preneoplastic lesion,cancer tissue, or cancer cells also can be used, provided that the cellsof the cell line have at least one or more antigenic determinant incommon with the antigens on the target cancer cells. Cancer tissues,cancer cells, cells infected with a cancer-causing agent, otherpreneoplastic cells, and cell lines of human origin can be used. Cancercells excised from the patient to whom ultimately the fusion proteinsultimately are to be administered can be particularly useful, althoughallogeneic cells also can be used. In some embodiments, a cancer cellcan be from an established tumor cell line such as, without limitation,an established non-small cell lung carcinoma (NSCLC), bladder cancer,melanoma, ovarian cancer, renal cell carcinoma, prostate carcinoma,sarcoma, breast carcinoma, squamous cell carcinoma, head and neckcarcinoma, hepatocellular carcinoma, pancreatic carcinoma, or coloncarcinoma cell line.

In various embodiments, the present fusion proteins allow for both thecostimulation T cell and the presentation of various tumor cellantigens. For instance, in some embodiments, the present vaccine proteinfusions (e.g., gp96 fusions) chaperone these various tumor antigens. Invarious embodiments, the tumor cells secrete a variety of antigens.Illustrative, but non-limiting, antigens that can be secreted are:MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (ADAbp), cyclophilin b, Colorectal associatedantigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, amll, Prostate SpecificAntigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zetachain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family oftumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,tyrosinase, p53, MUC family, HER2/neu, p2lras, RCAS1, α-fetoprotein,E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pme1117,PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC),fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides,viral products such as human papilloma virus proteins, Smad family oftumor antigens, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,SCP-1 CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70,CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, bladdercancer antigens such as ACTL8, ADAM22, ADAM23, ATAD2, ATAD2B, BIRC5,CASC5, CEP290, CEP55, CTAGE5, DCAF12, DDX5, FAM133A, IL13RA2, IMP3,KIAA0100, MAGEA11, MAGEA3, MAGEA6, MPHOSPH10, ODF2, ODF2L, OIP5, PBK,RQCD1, SPAG1, SPAG4, SPAG9, TMEFF1, TTK, and prostate cancer antigenssuch as PRAME, BIRC5, CEP55, ATAD2, ODF2, KIAA0100, SPAG9, GPATCH2,ATAD2B, CEP290, SPAG1, ODF2L, CTAGE5, DDX5, DCAF12, IMP3. In someembodiments, the antigens are human endogenous retroviral antigens.Illustrative antigens can also include antigens from human endogenousretroviruses which include, but are not limited to, epitopes derivedfrom at least a portion of Gag, at least a portion of Tat, at least aportion of Rev, a least a portion of Nef, and at least a portion ofgp160.

Further, in some embodiments, the present vaccine protein fusions (e.g.,gp96 fusions) provide for an adjuvant effect that further allows theimmune system of a patient, when used in the various methods describedherein, to be activated against a disease of interest.

Both prokaryotic and eukaryotic vectors can be used for expression ofthe vaccine protein (e.g., gp96-Ig) and T cell costimulatory fusionproteins in the methods provided herein. Prokaryotic vectors includeconstructs based on E. coli sequences (see, e.g., Makrides, MicrobiolRev 1996, 60:512-538). Non-limiting examples of regulatory regions thatcan be used for expression in E. coli include 1ac, trp, 1pp, phoA, recA,tac, T3, T7 and λP_(L). Non-limiting examples of prokaryotic expressionvectors may include the λgt vector series such as λgt11 (Huynh et al.,in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D.Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series(Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotichost-vector systems cannot perform much of the post-translationalprocessing of mammalian cells, however. Thus, eukaryotic host-vectorsystems may be particularly useful.

A variety of regulatory regions can be used for expression of thevaccine protein (e.g., gp96-Ig) and T cell costimulatory fusions inmammalian host cells. For example, the SV40 early and late promoters,the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcomavirus long terminal repeat (RSV-LTR) promoter can be used. Induciblepromoters that may be useful in mammalian cells include, withoutlimitation, promoters associated with the metallothionein II gene, mousemammary tumor virus glucocorticoid responsive long terminal repeats(MMTV-LTR), the β-interferon gene, and the hsp70 gene (see, Williams etal., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990,10:165-75). Heat shock promoters or stress promoters also may beadvantageous for driving expression of the fusion proteins inrecombinant host cells.

In an embodiment, the present invention contemplates the use ofinducible promoters capable of effecting high level of expressiontransiently in response to a cue. Illustrative inducible expressioncontrol regions include those comprising an inducible promoter that isstimulated with a cue such as a small molecule chemical compound.Particular examples can be found, for example, in U.S. Pat. Nos.5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which isincorporated herein by reference in its entirety.

Animal regulatory regions that exhibit tissue specificity and have beenutilized in transgenic animals also can be used in tumor cells of aparticular tissue type: the elastase I gene control region that isactive in pancreatic acinar cells (Swift et al., Cell 1984, 38:639-646;Ornitz et al., Cold Spring Harbor Symp Quant Biol 1986, 50:399-409; andMacDonald, Hepatology 1987, 7:425-515); the insulin gene control regionthat is active in pancreatic beta cells (Hanahan, Nature 1985,315:115-122), the immunoglobulin gene control region that is active inlymphoid cells (Grosschedl et al., Cell 1984, 38:647-658; Adames et al.,Nature 1985, 318:533-538; and Alexander et al., Mol Cell Biol 1987,7:1436-1444), the mouse mammary tumor virus control region that isactive in testicular, breast, lymphoid and mast cells (Leder et al.,Cell 1986, 45:485-495), the albumin gene control region that is activein liver (Pinkert et al., Genes Devel, 1987, 1:268-276), thealpha-fetoprotein gene control region that is active in liver (Krumlaufet al., Mol Cell Biol 1985, 5:1639-1648; and Hammer et al., Science1987, 235:53-58); the alpha 1-antitrypsin gene control region that isactive in liver (Kelsey et al., Genes Devel 1987, 1:161-171), thebeta-globin gene control region that is active in myeloid cells (Mogramet al., Nature 1985, 315:338-340; and Kollias et al., Cell 1986,46:89-94); the myelin basic protein gene control region that is activein oligodendrocyte cells in the brain (Readhead et al., Cell 1987,48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, Nature 1985, 314:283-286), and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., Science 1986, 234:1372-1378).

An expression vector also can include transcription enhancer elements,such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus,immunoglobulin genes, metallothionein, and β-actin (see, Bittner et al.,Meth Enzymol 1987, 153:516-544; and Gorman, Curr Op Biotechnol 1990,1:36-47). In addition, an expression vector can contain sequences thatpermit maintenance and replication of the vector in more than one typeof host cell, or integration of the vector into the host chromosome.Such sequences include, without limitation, to replication origins,autonomously replicating sequences (ARS), centromere DNA, and telomereDNA.

In addition, an expression vector can contain one or more selectable orscreenable marker genes for initially isolating, identifying, ortracking host cells that contain DNA encoding fusion proteins asdescribed herein. For long term, high yield production of gp96-Ig and Tcell costimulatory fusion proteins, stable expression in mammalian cellscan be useful. A number of selection systems can be used for mammaliancells. For example, the Herpes simplex virus thymidine kinase (Wigler etal., Cell 1977, 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalski and Szybalski, Proc Natl Acad Sci USA 1962, 48:2026), andadenine phosphoribosyltransferase (Lowy et al., Cell 1980, 22:817) genescan be employed in tk⁻, hgprt⁻, or aprt⁻cells, respectively. Inaddition, antimetabolite resistance can be used as the basis ofselection for dihydrofolate reductase (dhfr), which confers resistanceto methotrexate (Wigler et al., Proc Natl Acad Sci USA 1980, 77:3567;O'Hare et al., Proc Natl Acad Sci USA 1981, 78:1527); gpt, which confersresistance to mycophenolic acid (Mulligan and Berg, Proc Natl Acad SciUSA 1981, 78:2072); neomycin phosphotransferase (neo), which confersresistance to the aminoglycoside G-418 (Colberre-Garapin et al., J MolBiol 1981, 150:1); and hygromycin phosphotransferase (hyg), whichconfers resistance to hygromycin (Santerre et al., Gene 1984, 30:147).Other selectable markers such as histidinol and Zeocin™ also can beused.

Useful mammalian host cells include, without limitation, cells derivedfrom humans, monkeys, and rodents (see, for example, Kriegler in “GeneTransfer and Expression: A Laboratory Manual,” 1990, New York, Freeman &Co.). These include monkey kidney cell lines transformed by SV40 (e.g.,COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293,293-EBNA, or 293 cells subcloned for growth in suspension culture,Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells(e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (e.g., CHO,Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); mouse sertolicells (Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells(e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); Africangreen monkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); humancervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells(e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCCCRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells(e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT060562, ATCC CCL51). Illustrative cancer cell types for expressing thefusion proteins described herein include mouse fibroblast cell line,NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytomacell line, P815, mouse lymphoma cell line, EL4 and its ovalbumintransfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcomacell line, MC57, human small cell lung carcinoma cell lines, SCLC #2 andSCLC #7, human lung adenocarcinoma cell line, e.g., AD100, and humanprostate cancer cell line, e.g., PC-3.

A number of viral-based expression systems also can be used withmammalian cells to produce gp96-Ig and T cell costimulatory fusionproteins. Vectors using DNA virus backbones have been derived fromsimian virus 40 (SV40) (Hamer et al., Cell 1979, 17:725), adenovirus(Van Doren et al., Mol Cell Biol 1984, 4:1653), adeno-associated virus(McLaughlin et al., J Virol 1988, 62:1963), and bovine papillomas virus(Zinn et al., Proc Natl Acad Sci USA 1982, 79:4897). When an adenovirusis used as an expression vector, the donor DNA sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This fusion gene may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) can result in a recombinant virus that is viableand capable of expressing heterologous products in infected hosts. (See,e.g., Logan and Shenk, Proc Natl Acad Sci USA 1984, 81:3655-3659).

Bovine papillomavirus (BPV) can infect many higher vertebrates,including man, and its DNA replicates as an episome. A number of shuttlevectors have been developed for recombinant gene expression which existas stable, multicopy (20-300 copies/cell) extrachromosomal elements inmammalian cells. Typically, these vectors contain a segment of BPV DNA(the entire genome or a 69% transforming fragment), a promoter with abroad host range, a polyadenylation signal, splice signals, a selectablemarker, and “poisonless” plasmid sequences that allow the vector to bepropagated in E. coli. Following construction and amplification inbacteria, the expression gene constructs are transfected into culturedmammalian cells by, for example, calcium phosphate coprecipitation. Forthose host cells that do not manifest a transformed phenotype, selectionof transformants is achieved by use of a dominant selectable marker,such as histidinol and G418 resistance.

Alternatively, the vaccinia 7.5K promoter can be used. (See, e.g.,Mackett et al., Proc Natl Acad Sci USA 1982, 79:7415-7419; Mackett etal., J Virol 1984, 49:857-864; and Panicali et al., Proc Natl Acad SciUSA 1982, 79:4927-4931.) In cases where a human host cell is used,vectors based on the Epstein-Barr virus (EBV) origin (OriP) and EBVnuclear antigen 1 (EBNA-1; a trans-acting replication factor) can beused. Such vectors can be used with a broad range of human host cells,e.g., EBO-pCD (Spickofsky et al., DNA Prot Eng Tech 1990, 2:14-18); pDR2and λDR2 (available from Clontech Laboratories).

Gp96-Ig and T cell costimulatory fusion proteins also can be made withretrovirus-based expression systems. Retroviruses, such as Moloneymurine leukemia virus, can be used since most of the viral gene sequencecan be removed and replaced with exogenous coding sequence while themissing viral functions can be supplied in trans. In contrast totransfection, retroviruses can efficiently infect and transfer genes toa wide range of cell types including, for example, primary hematopoieticcells. Moreover, the host range for infection by a retroviral vector canbe manipulated by the choice of envelope used for vector packaging.

For example, a retroviral vector can comprise a 5′ long terminal repeat(LTR), a 3′ LTR, a packaging signal, a bacterial origin of replication,and a selectable marker. The gp96-Ig fusion protein coding sequence, forexample, can be inserted into a position between the 5′ LTR and 3′ LTR,such that transcription from the 5′ LTR promoter transcribes the clonedDNA. The 5′ LTR contains a promoter (e.g., an LTR promoter), an Rregion, a U5 region, and a primer binding site, in that order.Nucleotide sequences of these LTR elements are well known in the art. Aheterologous promoter as well as multiple drug selection markers alsocan be included in the expression vector to facilitate selection ofinfected cells. See, McLauchlin et al., Prog Nucleic Acid Res Mol Biol1990, 38:91-135; Morgenstern et al., Nucleic Acid Res 1990,18:3587-3596; Choulika et al., J Virol 1996, 70:1792-1798; Boesen etal., Biotherapy 1994, 6:291-302; Salmons and Gunzberg, Human Gene Ther1993, 4:129-141; and Grossman and Wilson, Curr Opin Genet Devel 1993,3:110-114.

Any of the cloning and expression vectors described herein may besynthesized and assembled from known DNA sequences using techniques thatare known in the art. The regulatory regions and enhancer elements canbe of a variety of origins, both natural and synthetic. Some vectors andhost cells may be obtained commercially. Non-limiting examples of usefulvectors are described in Appendix 5 of Current Protocols in MolecularBiology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience, which is incorporated herein by reference; and thecatalogs of commercial suppliers such as Clontech Laboratories,Stratagene Inc., and Invitrogen, Inc.

Methods of Treating

An expression vector as provided herein can be incorporated into acomposition for administration to a subject (e.g., a research animal ora mammal, such as a human, having a clinical condition such as cancer oran infection). For example, an expression vector can be administered toa subject for the treatment of cancer or infection. Thus, this documentprovides methods for treating clinical conditions such as cancer orinfection with the expression vectors provided herein. The infection canbe, for example, an acute infection or a chronic infection. In someembodiments, the infection can be an infection by hepatitis C virus,hepatitis B virus, human immunodeficiency virus, or malaria. The methodscan include administering to a subject an expression vector, a cellcontaining the expression vector, or a virus or virus-like particlecontaining the expression vector, under conditions wherein theprogression or a symptom of the clinical condition in the subject isreduced in a therapeutic manner.

In various embodiments, the present invention pertains to cancers and/ortumors; for example, the treatment or prevention of cancers and/ortumors. Cancers or tumors refer to an uncontrolled growth of cellsand/or abnormal increased cell survival and/or inhibition of apoptosiswhich interferes with the normal functioning of the bodily organs andsystems. Included are benign and malignant cancers, polyps, hyperplasia,as well as dormant tumors or micrometastases. Also, included are cellshaving abnormal proliferation that is not impeded by the immune system(e.g. virus infected cells). The cancer may be a primary cancer or ametastatic cancer. The primary cancer may be an area of cancer cells atan originating site that becomes clinically detectable, and may be aprimary tumor. In contrast, the metastatic cancer may be the spread of adisease from one organ or part to another non-adjacent organ or part.The metastatic cancer may be caused by a cancer cell that acquires theability to penetrate and infiltrate surrounding normal tissues in alocal area, forming a new tumor, which may be a local metastasis. Thecancer may also be caused by a cancer cell that acquires the ability topenetrate the walls of lymphatic and/or blood vessels, after which thecancer cell is able to circulate through the bloodstream (thereby beinga circulating tumor cell) to other sites and tissues in the body. Thecancer may be due to a process such as lymphatic or hematogeneousspread. The cancer may also be caused by a tumor cell that comes to restat another site, re-penetrates through the vessel or walls, continues tomultiply, and eventually forms another clinically detectable tumor. Thecancer may be this new tumor, which may be a metastatic (or secondary)tumor.

The cancer may be caused by tumor cells that have metastasized, whichmay be a secondary or metastatic tumor. The cells of the tumor may belike those in the original tumor. As an example, if a breast cancer orcolon cancer metastasizes to the liver, the secondary tumor, whilepresent in the liver, is made up of abnormal breast or colon cells, notof abnormal liver cells. The tumor in the liver may thus be a metastaticbreast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originatefrom melanoma, colon, breast, or prostate, and thus may be made up ofcells that were originally skin, colon, breast, or prostate,respectively. The cancer may also be a hematological malignancy, whichmay be lymphoma. The cancer may invade a tissue such as liver, lung,bladder, or intestinal.

Illustrative cancers that may be treated include, but are not limitedto, carcinomas, e.g. various subtypes, including, for example,adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, andtransitional cell carcinoma), sarcomas (including, for example, bone andsoft tissue), leukemias (including, for example, acute myeloid, acutelymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell),lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkinlymphomas, light chain, non-secretory, MGUS, and plasmacytomas), andcentral nervous system cancers (including, for example, brain (e.g.gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma),meningioma, pituitary adenoma, and neuromas, and spinal cord tumors(e.g. meningiomas and neurofibroma).

Representative cancers and/or tumors of the present invention include,but are not limited to, a basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and central nervous system cancer;breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

In some aspects, the present fusions are used to eliminate intracellularpathogens. In some aspects, the present fusions are used to treat one ormore infections. In some embodiments, the present fusion proteins areused in methods of treating viral infections (including, for example,HIV and HCV), parasitic infections (including, for example, malaria),and bacterial infections. In various embodiments, the infections induceimmunosuppression. For example, HIV infections often result inimmunosuppression in the infected subjects. Accordingly, as describedelsewhere herein, the treatment of such infections may involve, invarious embodiments, modulating the immune system with the presentfusion proteins to favor immune stimulation over immune inhibition.Alternatively, the present invention provides methods for treatinginfections that induce immunoactivation. For example, intestinalhelminth infections have been associated with chronic immune activation.In these embodiments, the treatment of such infections may involvemodulating the immune system with the present fusion proteins to favorimmune inhibition over immune stimulation.

In various embodiments, the present invention provides methods oftreating viral infections including, without limitation, acute orchronic viral infections, for example, of the respiratory tract, ofpapilloma virus infections, of herpes simplex virus (HSV) infection, ofhuman immunodeficiency virus (HIV) infection, and of viral infection ofinternal organs such as infection with hepatitis viruses. In someembodiments, the viral infection is caused by a virus of familyFlaviviridae. In some embodiments, the virus of family Flaviviridae isselected from Yellow Fever Virus, West Nile virus, Dengue virus,Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and HepatitisC Virus. In other embodiments, the viral infection is caused by a virusof family Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus.In other embodiments, the viral infection is caused by a member ofOrthomyxoviridae, e.g., an influenza virus. In other embodiments, theviral infection is caused by a member of Retroviridae, e.g., alentivirus. In other embodiments, the viral infection is caused by amember of Paramyxoviridae, e.g., respiratory syncytial virus, a humanparainfluenza virus, rubulavirus (e.g., mumps virus), measles virus, andhuman metapneumovirus. In other embodiments, the viral infection iscaused by a member of Bunyaviridae, e.g., hantavirus. In otherembodiments, the viral infection is caused by a member of Reoviridae,e.g., a rotavirus.

In various embodiments, the present invention provides methods oftreating parasitic infections such as protozoan or helminths infections.In some embodiments, the parasitic infection is by a protozoan parasite.In some embodiments, the oritiziab parasite is selected from intestinalprotozoa, tissue protozoa, or blood protozoa. Illustrative protozoanparasites include, but are not limited to, Entamoeba hystolytica,Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense,Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana,Leishmania braziliensis, Leishmania tropica, Leishmania donovani,Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonasmeleagridis. In some embodiments, the parasitic infection is by ahelminthic parasite such as nematodes (e.g., Adenophorea). In someembodiments, the parasite is selected from Secementea (e.g., Trichuristrichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostomaduodenale, Necator americanus, Strongyloides stercoralis, Wuchereriabancrofti, Dracunculus medinensis). In some embodiments, the parasite isselected from trematodes (e.g. blood flukes, liver flukes, intestinalflukes, and lung flukes). In some embodiments, the parasite is selectedfrom: Schistosoma mansoni, Schistosoma haematobium, Schistosomajaponicum, Fasciola hepatica, Fasciola giganfica, Heterophyesheterophyes, Paragonimus westermani. In some embodiments, the parasiteis selected from cestodes (e.g., Taenia solium, Taenia saginata,Hymenolepis nana, Echinococcus granulosus).

In various embodiments, the present invention provides methods oftreating bacterial infections. In various embodiments, the bacterialinfection is by a gram-positive bacteria, gram-negative bacteria,aerobic and/or anaerobic bacteria. In various embodiments, the bacteriais selected from, but not limited to, Staphylococcus, Lactobacillus,Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella,Pseudomonas, Acinetobacter, Mycobacterium, Proteus, Campylobacter,Citrobacter, Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium,Salmonella, Shigella, Serratia, Haemophilus, Brucella and otherorganisms. In some embodiments, the bacteria is selected from, but notlimited to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonasacidovorans, Pseudomonas alcaligenes, Pseudomonas pufida,Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonashydrophilia, Escherichia coli, Citrobacterfteundii, Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae,Klebsiella oxytoca, Serratia marcescens, Francisella tularensis,Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providenciaalcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacterbaumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus,Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis,Bordetella bronchisepfica, Haemophilus influenzae, Haemophilusparainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus,Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica,Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus,Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibriocholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeriamonocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella,Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroidesdistasonis, Bacteroides 3452A homology group, Bacteroides vulgatus,Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides eggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus. The expression vector(s),cells, or particles to be administered can be admixed, encapsulated,conjugated or otherwise associated with other molecules, molecularstructures, or mixtures of compounds such as, for example, liposomes,receptor or cell targeted molecules, or oral, topical or otherformulations for assisting in uptake, distribution and/or absorption. Insome cases, an expression vector can be contained within a cell that isadministered to a subject, or contained within a virus or virus-likeparticle. The vector, cell, or particle to be administered can be incombination with a pharmaceutically acceptable carrier.

This document therefore also provides compositions containing a vectoror a tumor cell or virus particle containing a vector encoding asecreted gp96-Ig fusion polypeptide and a T cell costimulatory fusionpolypeptide as described herein, in combination with a physiologicallyand pharmaceutically acceptable carrier. The physiologically andpharmaceutically acceptable carrier can be include any of the well-knowncomponents useful for immunization. The carrier can facilitate orenhance an immune response to an antigen administered in a vaccine. Thecell formulations can contain buffers to maintain a preferred pH range,salts or other components that present an antigen to an individual in acomposition that stimulates an immune response to the antigen. Thephysiologically acceptable carrier also can contain one or moreadjuvants that enhance the immune response to an antigen.Pharmaceutically acceptable carriers include, for example,pharmaceutically acceptable solvents, suspending agents, or any otherpharmacologically inert vehicles for delivering compounds to a subject.Pharmaceutically acceptable carriers can be liquid or solid, and can beselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, and other pertinent transportand chemical properties, when combined with one or more therapeuticcompounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include,without limitation: water, saline solution, binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose or dextrose and other sugars, gelatin, or calcium sulfate),lubricants (e.g., starch, polyethylene glycol, or sodium acetate),disintegrates (e.g., starch or sodium starch glycolate), and wettingagents (e.g., sodium lauryl sulfate). Compositions can be formulated forsubcutaneous, intramuscular, or intradermal administration, or in anymanner acceptable for immunization.

An adjuvant refers to a substance which, when added to an immunogenicagent such as a tumor cell expressing secreted vaccine protein (e.g.,gp96-Ig) and T cell costimulatory fusion polypeptides, nonspecificallyenhances or potentiates an immune response to the agent in the recipienthost upon exposure to the mixture. Adjuvants can include, for example,oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts),liposomes and microparticles, such as, polysytrene, starch,polyphosphazene and polylactide/polyglycosides.

Adjuvants can also include, for example, squalene mixtures (SAF-I),muramyl peptide, saponin derivatives, mycobacterium cell wallpreparations, monophosphoryl lipid A, mycolic acid derivatives, nonionicblock copolymer surfactants, Quil A, cholera toxin B subunit,polyphosphazene and derivatives, and immunostimulating complexes(ISCOMs) such as those described by Takahashi et al., Nature 1990,344:873-875. For veterinary use and for production of antibodies inanimals, mitogenic components of Freund's adjuvant (both complete andincomplete) can be used. In humans, Incomplete Freund's Adjuvant (IFA)is a useful adjuvant. Various appropriate adjuvants are well known inthe art (see, for example, Warren and Chedid, CRC Critical Reviews inImmunology 1988, 8:83; and Allison and Byars, in Vaccines: NewApproaches to Immunological Problems, 1992, Ellis, ed.,Butterworth-Heinemann, Boston). Additional adjuvants include, forexample, bacille Calmett-Guerin (BCG), DETOX (containing cell wallskeleton of Mycobacterium phlei (CWS) and monophosphoryl lipid A fromSalmonella minnesota (MPL)), and the like (see, for example, Hoover etal., J Clin Oncol 1993, 11:390; and Woodlock et al., J Immunother 1999,22:251-259).

In some embodiments, a vector can be administered to a subject one ormore times (e.g., once, twice, two to four times, three to five times,five to eight times, six to ten times, eight to 12 times, or more than12 times). A vector as provided herein can be administered one or moretimes per day, one or more times per week, every other week, one or moretimes per month, once every two to three months, once every three to sixmonths, or once every six to 12 months. A vector can be administeredover any suitable period of time, such as a period from about 1 day toabout 12 months. In some embodiments, for example, the period ofadministration can be from about 1 day to 90 days; from about 1 day to60 days; from about 1 day to 30 days; from about 1 day to 20 days; fromabout 1 day to 10 days; from about 1 day to 7 days. In some embodiments,the period of administration can be from about 1 week to 50 weeks; fromabout 1 week to 50 weeks; from about 1 week to 40 weeks; from about 1week to 30 weeks; from about 1 week to 24 weeks; from about 1 week to 20weeks; from about 1 week to 16 weeks; from about 1 week to 12 weeks;from about 1 week to 8 weeks; from about 1 week to 4 weeks; from about 1week to 3 weeks; from about 1 week to 2 weeks; from about 2 weeks to 3weeks; from about 2 weeks to 4 weeks; from about 2 weeks to 6 weeks;from about 2 weeks to 8 weeks; from about 3 weeks to 8 weeks; from about3 weeks to 12 weeks; or from about 4 weeks to 20 weeks.

In some embodiments, after an initial dose (sometimes referred to as a“priming” dose) of a vector has been administered and a maximalantigen-specific immune response has been achieved, one or more boostingdoses of a vector as provided herein can be administered. For example, aboosting dose can be administered about 10 to 30 days, about 15 to 35days, about 20 to 40 days, about 25 to 45 days, or about 30 to 50 daysafter a priming dose.

In some embodiments, the methods provided herein can be used forcontrolling solid tumor growth (e.g., breast, prostate, melanoma, renal,colon, or cervical tumor growth) and/or metastasis. The methods caninclude administering an effective amount of an expression vector asdescribed herein to a subject in need thereof. In some embodiments, thesubject is a mammal (e.g., a human).

The vectors and methods provided herein can be useful for stimulating animmune response against a tumor. Such immune response is useful intreating or alleviating a sign or symptom associated with the tumor. Asused herein, by “treating” is meant reducing, preventing, and/orreversing the symptoms in the individual to which a vector as describedherein has been administered, as compared to the symptoms of anindividual not being treated. A practitioner will appreciate that themethods described herein are to be used in concomitance with continuousclinical evaluations by a skilled practitioner (physician orveterinarian) to determine subsequent therapy. Such evaluations will aidand inform in evaluating whether to increase, reduce, or continue aparticular treatment dose, mode of administration, etc.

The methods provided herein can thus be used to treat a tumor,including, for example, a cancer. The methods can be used, for example,to inhibit the growth of a tumor by preventing further tumor growth, byslowing tumor growth, or by causing tumor regression. Thus, the methodscan be used, for example, to treat a cancer such as a lung cancer. Itwill be understood that the subject to which a compound is administeredneed not suffer from a specific traumatic state. Indeed, the vectorsdescribed herein may be administered prophylactically, prior todevelopment of symptoms (e.g., a patient in remission from cancer). Theterms “therapeutic” and “therapeutically,” and permutations of theseterms, are used to encompass therapeutic, palliative, and prophylacticuses. Thus, as used herein, by “treating or alleviating the symptoms” ismeant reducing, preventing, and/or reversing the symptoms of theindividual to which a therapeutically effective amount of a compositionhas been administered, as compared to the symptoms of an individualreceiving no such administration.

As used herein, the terms “effective amount” and “therapeuticallyeffective amount” refer to an amount sufficient to provide the desiredtherapeutic (e.g., anti-cancer, anti-tumor, or anti-infection) effect ina subject (e.g., a human diagnosed as having cancer or an infection).Anti-tumor and anti-cancer effects include, without limitation,modulation of tumor growth (e.g., tumor growth delay), tumor size, ormetastasis, the reduction of toxicity and side effects associated with aparticular anti-cancer agent, the amelioration or minimization of theclinical impairment or symptoms of cancer, extending the survival of thesubject beyond that which would otherwise be expected in the absence ofsuch treatment, and the prevention of tumor growth in an animal lackingtumor formation prior to administration, i.e., prophylacticadministration. In some embodiments, administration of an effectiveamount of a vector or a composition, cell, or virus particle containingthe vector can increase the activation or proliferation of tumor antigenspecific T cells in a subject. For example, the activation orproliferation of tumor antigen specific T cells in the subject can be isincreased by at least 10 percent (e.g., at least 25 percent, at least 50percent, or at least 75 percent) as compared to the level of activationor proliferation of tumor antigen specific T cells in the subject priorto the administration.

Anti-infection effects include, for example, a reduction in the numberof infective agents (e.g., viruses or bacteria). When the clinicalcondition in the subject to be treated is an infection, administrationof a vector as provided herein can stimulate the activation orproliferation of pathogenic antigen specific T cells in the subject. Forexample, administration of the vector can lead to activation ofantigen-specific T cells in the subject to a level great than thatachieved by 96-Ig vaccination alone.

One of skill will appreciate that an effective amount of a vector may belowered or increased by fine tuning and/or by administering more thanone dose (e.g., by concomitant administration of two differentgenetically modified tumor cells containing the vector), or byadministering a vector with another agent (e.g., an antagonist of PD-1)to enhance the therapeutic effect (e.g., synergistically). This documenttherefore provides a method for tailoring the administration/treatmentto the particular exigencies specific to a given mammal Therapeuticallyeffective amounts can be determined by, for example, starting atrelatively low amounts and using step-wise increments with concurrentevaluation of beneficial effects. The methods provided herein thus canbe used alone or in combination with other well-known tumor therapies,to treat a patient having a tumor. One skilled in the art will readilyunderstand advantageous uses of the vectors and methods provided herein,for example, in prolonging the life expectancy of a cancer patientand/or improving the quality of life of a cancer patient (e.g., a lungcancer patient).

Combination Therapies and Conjugation

In some embodiments, the invention provides for methods that furthercomprise administering an additional agent to a subject. In someembodiments, the invention pertains to co-administration and/orco-formulation.

In some embodiments, administration of vaccine protein (e.g., gp96-Ig)and one or more costimulatory molecules act synergistically whenco-administered with another agent and is administered at doses that arelower than the doses commonly employed when such agents are used asmonotherapy.

In some embodiments, inclusive of, without limitation, cancerapplications, the present invention pertains to chemotherapeutic agentsas additional agents. Examples of chemotherapeutic agents include, butare not limited to, alkylating agents such as thiotepa and CYTOXANcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomy sins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In some embodiments, inclusive of, without limitation, infectiousdisease applications, the present invention pertains to anti-infectivesas additional agents. In some embodiments, the anti-infective is ananti-viral agent including, but not limited to, Abacavir, Acyclovir,Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine,Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine,Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. In someembodiments, the anti-infective is an anti-bacterial agent including,but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime,cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin,cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro,Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics(tetracycline, minocycline, oxytetracycline, and doxycycline);penicillin antibiotics (amoxicillin, ampicillin, penicillin V,dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactamantibiotics (aztreonam); and carbapenem antibiotics (ertapenem,doripenem, imipenem/cilastatin, and meropenem). In some embodiments, theanti-infectives include anti-malarial agents (e.g., chloroquine,quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine,atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole,tinidazole, ivermectin, pyrantel pamoate, and albendazole.

Other additional agents are described elsewhere herein, including theblocking antibodies targeted to an immune “checkpoint” molecules.

Subjects and/or Animals

The methods described herein are intended for use with any subject thatmay experience the benefits of these methods. Thus, “subjects,”“patients,” and “individuals” (used interchangeably) include humans aswell as non-human subjects, particularly domesticated animals.

In some embodiments, the subject and/or animal is a mammal, e.g., ahuman, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep,or non-human primate, such as a monkey, chimpanzee, or baboon. In otherembodiments, the subject and/or animal is a non-mammal, such, forexample, a zebrafish. In some embodiments, the subject and/or animal maycomprise fluorescently-tagged cells (with e.g. GFP). In someembodiments, the subject and/or animal is a transgenic animal comprisinga fluorescent cell.

In some embodiments, the subject and/or animal is a human In someembodiments, the human is a pediatric human In other embodiments, thehuman is an adult human In other embodiments, the human is a geriatrichuman In other embodiments, the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0months to about 6 months old, from about 6 to about 12 months old, fromabout 6 to about 18 months old, from about 18 to about 36 months old,from about 1 to about 5 years old, from about 5 to about 10 years old,from about 10 to about 15 years old, from about 15 to about 20 yearsold, from about 20 to about 25 years old, from about 25 to about 30years old, from about 30 to about 35 years old, from about 35 to about40 years old, from about 40 to about 45 years old, from about 45 toabout 50 years old, from about 50 to about 55 years old, from about 55to about 60 years old, from about 60 to about 65 years old, from about65 to about 70 years old, from about 70 to about 75 years old, fromabout 75 to about 80 years old, from about 80 to about 85 years old,from about 85 to about 90 years old, from about 90 to about 95 years oldor from about 95 to about 100 years old.

In other embodiments, the subject is a non-human animal, and thereforethe invention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal In certain embodiments, thesubject is a human cancer patient that cannot receive chemotherapy, e.g.the patient is unresponsive to chemotherapy or too ill to have asuitable therapeutic window for chemotherapy (e.g. experiencing too manydose- or regimen-limiting side effects). In certain embodiments, thesubject is a human cancer patient having advanced and/or metastaticdisease.

As used herein, an “allogeneic cell” refers to a cell that is notderived from the individual to which the cell is to be administered,that is, has a different genetic constitution than the individual. Anallogeneic cell is generally obtained from the same species as theindividual to which the cell is to be administered. For example, theallogeneic cell can be a human cell, as disclosed herein, foradministering to a human patient such as a cancer patient. As usedherein, an “allogeneic tumor cell” refers to a tumor cell that is notderived from the individual to which the allogeneic cell is to beadministered. Generally, the allogeneic tumor cell expresses one or moretumor antigens that can stimulate an immune response against a tumor inan individual to which the cell is to be administered. As used herein,an “allogeneic cancer cell,” for example, a lung cancer cell, refers toa cancer cell that is not derived from the individual to which theallogeneic cell is to be administered.

As used herein, a “genetically modified cell” refers to a cell that hasbeen genetically modified to express an exogenous nucleic acid, forexample, by transfection or transduction.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined.

As used herein, the singular forms “a,” “an” and “the” specifically alsoencompass the plural forms of the terms to which they refer, unless thecontent clearly dictates otherwise. As used herein, unless specificallyindicated otherwise, the word “or “is used in the “inclusive” sense of“and/or” and not the “exclusive” sense of either/or.” In thespecification and the appended claims, the singular forms include pluralreferents unless the context clearly dictates otherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%. As used in this specification,whether in a transitional phrase or in the body of the claim, the terms“comprise (s)” and “comprising” are to be interpreted as having anopen-ended meaning. That is, the terms are to be interpretedsynonymously with the phrases “having at least” or “including at least”.When used in the context of a process, the term “comprising” means thatthe process includes at least the recited steps, but may includeadditional steps. When used in the context of a compound or composition,the term “comprising” means that the compound or composition includes atleast the recited features or components, but may also includeadditional features or components.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Vector Engineered Immunotherapy Incorporating gp96-Igand T cell Costimulatory fusion Proteins Elicits a SuperiorAntigen-Specific CD8+ T cell Response

Secretable heat-shock protein gp96-Ig based allogeneic cellular vaccinescan achieve high frequency polyclonal CD8+ T cell responses tofemtomolar concentrations of tumor antigens through antigencross-priming in vivo. Multiple immunosuppressive mechanisms evolved byestablished tumors can dampen the activity of this vaccine approach. Asdescribed below, a systematic comparison of PD-1, PD-L1, CTLA-4, andLAG-3 blocking antibodies in mouse models of long-established B16-F10melanoma demonstrated a superior combination between gp96-Ig vaccinationand PD-1 blockade as compared to other checkpoints. Triple combinationsof gp96-Ig vaccination, PD-1 blockade, and T cell costimulation usingOX40, ICOS, or 4-1BB agonists provided a synergistic anti-tumor benefit.

A 96-Ig expression vector was re-engineered to simultaneously co-expressICOSL-Ig, 4-1BBL-Ig, or OX40L-Ig, thus providing a costimulatory benefitwithout the need for additional antibody therapy. As described below,co-secretion of gp96-Ig and these costimulatory fusion proteins inallogeneic cell lines resulted in enhanced activation ofantigen-specific CD8+ T cells. Thus, combination immunotherapy can beachieved by vector re-engineering, obviating the need forvaccine/antibody/fusion protein regimens, and importantly may limit bothcost of therapy and the risk of systemic toxicity.

Example 2—Vaccine+Costimulator Vector Re-Engineering

A vector re-engineering strategy was employed to incorporate vaccine andT cell costimulatory fusion proteins into a single vector. Specifically,the original gp96-Ig vector was re-engineered to generate a cell-basedcombination IO product that secretes both the 96-Ig fusion protein andvarious T cell costimulatory fusion proteins (FIGS. 1 and 2). Thecombined local secretion of vaccine and costimulatory fusion protein(FIG. 3) can activate tumor antigen specific T cells, and is anticipatedto enhance antigen-specific immunity with limited cost and systemictoxicity, particularly when combined with administration of an agent(e.g., an antibody against PD-1) that inhibits immunosuppressivemolecules produced by tumor cells.

Example 3—In Vivo Studies of ImPACT Vs. ComPACT

Materials and Methods

Cell culture and vaccine cell line generation: 3T3 cells were maintainedin IMDM with glutamine and 10% Bovine Growth Serum (BGS) at 37° C. in 5%CO₂. A 3T3-Ovalbumin-Hygro parental cell line was established usinghygromycin resistant plasmid backbone pcDNA3.1 encoding chickenovalbumin (Ova) through nucleofection with the 4D-NUCLEOFECTOR™ and CellLine NUCLEOFECTOR™ Kit SE (Lonza) according to the manufacturer'sdirections. Single cell clones secreting high-level Ova were screened byELISA and used to generate 3T3-Ova-Gp96-Ig (ImPACT) and3T3-Ova-Gp96-Ig/OX40L-Fc (ComPACT) through nucleofection of G418resistant plasmid pB45 encoding either murine Gp96-Ig or Gp96-Ig and theextracellular domain of OX40L-Fc, respectively. Again, single cellclones of both ImPACT and ComPACT were generated through antibioticselection and clones secreting similar levels of mouse IgG were screenedfurther and used for subsequent analysis. OX40L mRNA expression wasconfirmed by qRT-PCR and protein levels were assessed by western blot.

CT26 cells were maintained in IMDM with glutamine and 10% Fetal BovineSerum at 37° C. in 5% CO₂. CT26 versions of ImPACT (CT26-Gp96-Ig) andComPACT (CT26-Gp96-Ig/OX40L-Fc) were generated using the same expressionplasmids as above, however transfected into the CT26 cell line usingEFFECTENE® Transfection Reagents (Qiagen) according to manufacturer'sdirections. Single cell clones were isolated under antibiotic selectionand screened for mouse IgG secretion by ELISA. OX40L mRNA expression wasconfirmed by qRT-PCR.

B16.F10 cell lines were first established by generating an ova parentalclone (B16.F10-ova: as described above for 3T3 cells). Then, B16.F10-ovaversions of ImPACT (B16.F10-ova-96-Ig) and ComPACT(B16.F10-ova-gp96-Ig/Fc-OX40L) were again transfected with the identicalplasmids as described above, and selected for high-level gp96-Igsecretion.

Mouse models, OT-I/OT-II transfer and analysis: Antigen specific CD8 Tcells were isolated from the spleens of OT-I/EGFP mice, carrying the Tcell receptor transgenes TCRα-V2 and TCRβ-V5, that recognize ovalbuminresidues 257-264 during H2K^(b) MHC class I antigen cross-presentation.Antigen specific CD4 T cells were isolated from the spleens of OT-IImice, expressing the mouse α- and β-chain T cell receptor that iscomplementary with the CD4 co-receptor and is specific for chickenovalbumin residues 323-339 during I-A^(b) MHC class II antigencross-presentation.

Briefly, mice were sacrificed through CO₂ asphyxiation followed bycervical dislocation, and the spleen was dissected into sterile PBS+2 mMEDTA. Splenocytes were dissociated from the tissue and passed through a100 μM strainer. Cells were pelleted at 1,200 RPM for 5 minutes and redblood cells were lysed by adding 5 mL 1×ACK lysis buffer (150 mM NH₄Cl,10 mM KHCO₃ and 1 mM EDTA) for 1-2 minutes at room temperature.Following lysis, an equal volume of 1×PBS was added and the cells wereagain pelleted at 1,200 RPM for 5 minutes. OT-I (CD8) and OT-II (CD4)were isolated from total splenocytes using CD4 and CD8 isolation kitsfrom StemCell Technologies, according to the manufacturer's directions.OT-I (0.5×10⁶ cells per mouse) and OT-II (1×10⁶ cells per mouse) weretransferred via intravenous (IV) tail vein injections to mice transgenicfor FOXP3-RFP (to track regulatory T cells: Tregs). The IV injection daycorresponded to experimental day −1.

On days 0 and 35 (in the case of boosted mice), mice were eitheruntreated, vaccinated with the 3T3-Ova parental clone as a control,vaccinated with ImPACT (alone or in combination with 100 μg of agonistantibodies to ICOS (BioLegend #313512), 4-1BB (3H3 antibody, Bio-X-Cell)or OX40 (OX86 antibody, Bio-X-Cell), or vaccinated with ComPACT.Vaccinations consisted of 1×10⁶ cells and were administered byintraperitoneal injection (IP). Lymphocytes harvested from peripheralblood were analyzed by flow cytometry throughout the time-course.

CT26 Tumor Model and Analysis: For CT26 tumor studies, BALB/C mice wereinoculated with either 2×10⁵ or 5×10⁵ tumor cells via subcutaneousinjection into the rear flank, indicating day 0. For B16.F10-ovastudies, C57BL/6 mice were inoculated with 5×10⁵ tumor cells into therear flank, indicating day 0. On vaccination days, tumor bearing micewere either untreated or vaccinated with mitomycin-C (Sigma) treatedImPACT, ImPACT+100 mg anti-OX86 (referred to as OX40(ab) throughout) orComPACT cells. Tumor area (mm²) and overall survival was assessedthroughout the time course. 30-day survival criteria included totaltumor area less than 175 mm² with no sign of tumor ulceration. Completeresponders, in which tumors established and were subsequently rejectedfollowing treatment, are listed in FIG. 12D, and FIG. 11E. A cohort ofmice inoculated with 2×10⁵ cells was sacrificed 11 days after tumorinoculation. Tumors were excised from these mice, trypsinized at 37° C.for 10 minutes, dissociated, and passed through a 100 μM cell strainer.Cells were pelleted, red blood cells were lysed (as described above),and RNA was isolated, reverse transcribed, and analyzed by qPCR (seebelow). A cohort of CT26 experimental mice was euthanized on day 12 forAH1-tetramer analysis in splenocytes and genetic analysis of tumortissue. Tumors were excised from these mice, trypsinized at 37° C. for10-15 minutes, dissociated and homogenized through a 100 mM strainer.Cells were pelleted and processed for RNA isolation (see below).

Flow Cytometry: Flow cytometry and cell sorting was performed on theSony SH800. For extracellular staining, cell pellets were resuspended in1×PBS buffer containing 1% bovine serum albumin (BSA), 0.02% sodiumazide, and 2 mM EDTA, and the appropriate antibodies and incubated onice in the dark for 30 minutes. Cells were then washed in flow cytometrybuffer, resuspended and then analyzed. For intracellular staining, cellswere fixed and permeabilized using the FOXP3 Fix/Perm kit fromBioLegend, stained as described above, washed in flow cytometry buffer,resuspended and then analyzed. Antibodies used were PE/Cy7-CD4 (Sony,1102640), AF700-CD8a (Sony, 1103650), APC-TCR Vβ5.1,5.2 (Sony, 1297530),PacificBlue-TCR Vα2 (Sony, 1239080), APC-KLRG1 (BioLegend, 138412),BV421-CD44 (BioLegend, 103039), BV605-CD127 (BioLegend, 135025),APC-Ki67 (BioLegend, 652406), PE/Cy7-IFNγ (Biolegend, 505826), andBV421-IL2 (BioLegend, 503825).

ELISAs: Standard ELISA conditions were set such that 1×10⁶ cells wereplated in 1 mL of culture media and the supernatant analyzed after 24hours. High-binding ELISA plates were coated with 10 μg/mL mouse IgG(Jackson Laboratories #115-005-062) in sodium bicarbonate buffer. Coatedplates were incubated over night at 4° C. The following morning, plateswere washed 3 times with TBS-T (50 mM Tris, 150 mM NaCl and 0.05% Tween20), blocked for 1 hour with Casein Blocking Buffer (Sigma) and againwashed 3 times with TBS-T. To the plates, 50 μL of cell supernatantsalong with an 11-point mouse IgG standard set of samples were added tothe coated ELISA plates and incubated at room temperature for 1 hour.Plates were washed 3 times with TBS-T and 50 μL of detection antibody(Jackson Laboratories #115-035-071) was added, and incubated for 1 hourat room temperature in the dark. Plates were washed 3 times with TBS-Tand 100 μL of SUREBLUE™ TMB Microwell Peroxidase Substrate (KPL) wasadded to each well and allowed to incubate at room temperature for 20minutes in the dark. To stop the reaction, 100 μL of sulfuric acid wasadded to each well and plates were read immediately on a BioTek platereader. Samples were run at least in triplicate at multiple dilutions.

RNA isolation and qRT-PCR: Total RNA was prepared using RNeasy andRNeasy Micro kits (Qiagen) according to the manufacturer'srecommendations, including on-column DNase treatment. A total of 1 μg(using RNeasy) or 100 ng (using RNeasy Micro) was used to synthesizecDNA with the First-strand cDNA synthesis kit from OriGene. qPCR wasperformed using KAPA SYBR FAST, SYBR green master mix (Kapa Biosystems)and then analyzed on a Roche Lightcycler. Values were normalized to 18SmRNA and represent the average±standard error of the mean (SEM) for aminimum of 3 biological replicates, all run in triplicate. Primersequences used were:

IFN-gamma: (SEQ ID NO: 14) F: 5′-CTGCCACGGCACAGTCATTG-3′ (SEQ ID NO: 15)R: 5′-gccagttcctccagatatcc-3′ TNF-alpha: (SEQ ID NO: 16) F:5′-CCACGCTCTTCTGTCTACTG-3′ (SEQ ID NO: 17) R:3′-gccatagaactgatgagaggg-3′ granzyme-B (SEQ ID NO: 18) F:5′-CTACTGCTGACCTTGTCTCTG-3′ (SEQ ID NO: 19) R:3′-agtaaggccatgtagggtcg-3′ IL-2 (SEQ ID NO: 20) F:5′-CTGCGGCATGTTCTGGATTTGACT-3′ (SEQ ID NO: 21) R:5′-AGTCCACCACAGTTGCTGACTCAT-3′ perforin-1 (SEQ ID NO: 22) F:5′-GACACAGTAGAGTGTCGCATG-3′ (SEQ ID NO: 23) R:5′-aagcatgctctgtggagctg-3′ beta-actin (SEQ ID NO: 24) F:5′-aaggccaaccgtgaaaagat-3′ (SEQ ID NO: 25) R: 5′-gtggtacgaccagaggcatac3′

Western Blot Analysis: ImPACT and ComPACT cells were treated for 16hours with Brefeldin-A to inhibit protein transport and secretion. Cellswere then lysed in RIPA buffer (25 mM Tris-HCL, 150 mM NaCl, 1% NP-40,1% NaDeoxycholate, and 0.1% SDS), containing 1× complete proteaseinhibitor cocktail (Roche) for 10 minutes on ice. Protein concentrationwas determined using DC Protein Assay kit (Bio-Rad) and 20 μg of proteinwas probed. Antibodies were: CD252 (OX40L, Abcam #ab156285, 1:1000dilution), histone H3 (Active Motif #61278, 1:10,000), histone H4(Active Motif #61300, 1:10,000), and beta actin (Abcam #ab8226,1:10,000).

LEGENDplex Cytokine Analysis: Experimental mice were euthanized throughCO₂ asphyxiation and cervical dislocation and whole blood was collectedvia cardiac puncture. Red blood cells were allowed to settle by gravityfor 1 hour at room temperature and the remaining cells were pelleted at1,200 RPM for 5 minutes. Serum was then transferred to a new 1.5 mLeppendorf tube. Cytokine analysis was performed using the LEGENDPLEX™Cytokine Analysis kit (BioLegend) according to manufacturerrecommendations and analyzed on the Sony SH800.

Statistical Analysis: Experimental replicates (N) are shown in thefigures. Unless noted otherwise, values plotted represent the mean froma minimum of 3 distinct experiments and error is SEM. Statisticalsignificance (p-value) was determined using unpaired parametric t-testswith Welch's correction. Significant p-values are labeled with anasterisk (*), and the corresponding p-value is labeled in each figure.

Results

Many new trials will investigate whether adding a therapeutic vaccine orT cell costimulatory antibody is an effective strategy to increase theproportion of responding patients and the durability of clinicalresponses. The implementation of such a strategy is limited by severalfactors, including an incomplete understanding of which agents mayprovide synergistic benefit, whether to toxicities of such combinationswill be tolerable and eventually how the healthcare system will managesuch combinations.

To investigate the potential synergy between a vaccine and individual Tcell costimulatory molecules, a series of head-to-head studies wasperformed in pre-clinical mouse models. Using a cell-based vaccineexpressing a modified secretable 96-Ig fusion protein (FIG. 4A), studieswere conducted to investigate whether co-administration of agonisticantibodies targeting OX40, 4-1BB, or ICOS would provide furthercostimulation of antigen-specific CD8+ T cells (FIGS. 5A-5) Immunizationof C57BL/6 mice that were adoptively transferred with ovalbumin-specificCD8+ T cells (OT-I) with a 3T3-ova-gp96-Ig vaccine led to proliferationof OT-I cells to 10% of peripheral blood CD8+ T cells. This responsecould be doubled by additional administration of OX40 agonistantibodies, but not 4-1BB or ICOS co-stimulatory antibodies (FIG. 5D).

T cell costimulation by OX40L is triggered by local inflammation in aspatially restricted microenvironment by antigen presenting cells overthe course of 2-5 days. Administration of OX40 receptor agonistantibodies provides systemic costimulation that can persist for severalweeks. Since vaccines are typically administered locally, experimentswere performed to determine whether an OX40L fusion protein (Fc-OX40L)could be co-expressed in the second cassette of the gp96-Ig containingplasmid as a strategy to both limit systemic co-stimulation and enablecombination immunotherapy with a single compound (FIG. 6A). As proof ofconcept, a 3T3 cell co-expressing soluble ovalbumin and either gp96-Igalone (“ImPACT”) or gp96-Ig together with Fc-OX40L, ICOSL, or 4-1BBL wasgenerated. These cell lines were stably selected to secrete similaramounts of both ova and gp96-Ig (FIGS. 7A and 7B). Expression ofFc-OX40L, ICOSL, or 4-1BBL was evaluated by RT-PCR and Western blotting(FIGS. 7C and 7D), and shown to be functionally active in cell culturesupernatants by an IL-2 secretion assay from primary splenocytes.

The in vivo activity of ImPACT either alone or in combination with OX40agonist antibodies was compared to ComPACT using the OT-I modeldescribed in FIG. 5. Distinct cell lines were used in this experimentbecause the co-transfections described above were not possible with theneomycin resistance cassette expressing ova in FIG. 5. Since ComPACT wasadministered locally, one might not have expected the dramatic primingand boosting effects vs. the ImPACT combination with the OX40 agonistantibody, which was administered systemically. As shown in FIGS. 6B and6C, however, ComPACT immunization provided surprisingly andsignificantly improved proliferation of OT-I cells following primaryimmunization either with ImPACT alone or in combination with OX40agonist antibodies. The peak expansion in the peripheral blood wasincreased on day 5 with ComPACT, but more importantly, so was theduration of the response from days 6-20.

The memory response to OX40 agonistic antibodies in combination withvaccination is relatively weak within the antigen specific CD8compartment. The boost response was evaluated by re-immunizing mice onday 35 after the primary immunization (FIG. 6C). While the combinationof OX40 agonist antibodies provided a relatively weak boost of the OT-Iresponse, ComPACT treated mice demonstrated a boost response that nearlymatched the magnitude of the primary response (FIG. 6C). Flow cytometricanalysis of splenocytes and peritoneal cells from mice receivingComPACT, revealed a marked increase in CD127⁺KLRG1⁻ cells compared tothe other groups, indicating an increase in memory precursor cells (FIG.8B). This effect was observed with various ComPACTS, including ComPACT(OX40L), ComPACT (ICOSL) and ComPACT (4-1BBL) but not with OX40 agonistantibody treatment. The various ComPACTs did not induce an increase inshort-lived effector cells (CD127⁻KLRG1⁺, FIG. 8B), as did OX40 agonistantibody treatment. The ComPacts, however, did increase memory T cells(CD127⁺KLRG1⁺) within the spleen (FIG. 8B). These data indicate thatlocal administration of an OX40L, ICOSL, or 4-1BBL agonist fusionproteins significantly increased both the primary and the boost responsein the antigen-specific CD8 compartment, which is correlated with anincrease in memory precursor cells and a prolonged contraction phasefollowing priming In addition, these data also revealed a novel andunexpected mechanism of action for ComPACT treated mice in comparison toImPACT +/−OX40 agonist antibody.

It was possible that the reason for increased primary and boostresponses in the antigen-specific CD8 compartment with locally providedOX40L was due to decreased off-target activation provided by systemicadministration of OX40 agonist antibodies. To test this hypothesis,peritoneal cells, splenocytes and tumor draining lymph node (TDLN) cellswere isolated on day 8 from mice that were immunized with ImPACT+/−OX40agonist antibody or ComPACT and analyzed by flow cytometry andquantitative RT-PCR (qRT-PCR) to distinguish between off-target immuneactivation and an antigen-specific response. Analysis of peritonealcells isolated on day 8 following primary immunization indicatedincreased numbers of total mononuclear, OT-I, and OT-II cells in ComPACTtreated mice, but also increased numbers of total CD4 cells in micetreated with OX40 agonist antibodies (FIG. 8A). Increased levels oftotal CD4+ cells and FOXP3+ regulatory T cells (Treg) were detected inthe peritoneal cavity, spleen and TDLN in mice treated with OX40 agonistantibodies (FIGS. 8A and 8E). In contrast, ComPACT treated micespecifically amplified antigen-specific OT-I (CD8+) and OT-II (CD4+)cells with no apparent stimulation of Treg cells (FIGS. 8A and 8E)Similar findings also were observed in the spleen and lymph nodes,indicating systemic expansion of total CD4 cells as well asantigen-specific CD4 cells (FIGS. 9A and 9B). CD4+FoxP3+ regulatory Tcells (Treg) also were increased for OX40 agonist, but not ComPACTtreated animals. Serum cytokine analysis further demonstrated a systemicincrease in IFNγ, TNFα, IL-5 and IL-6 in mice treated with OX40 agonistantibodies (FIG. 8C). To investigate the cellular source of the systemiccytokine increase, RT-PCR was performed on either total CD8+ cells orOT-I cells on day 8 following immunization. ComPACT treated mice showedan increase in IFNγ, TNFα and granzyme-B that was isolated to the OT-Ipopulation, whereas mice treated with OX40 agonist antibodies showed anincrease in both the OT-I and the total CD8 population (FIG. 8D).

These data indicate that OX40L fusion proteins can be locally providedby stable transfection of a plasmid co-expressing a heat shock proteingp96-Ig based vaccine. Initial feasibility related to whether sufficientconcentrations of Fc-OX40L were secreted to provide costimulationdemonstrated that this was achievable, and surprisingly, more effectivethan systemic administration of OX40 agonist antibodies. Thecostimulated OT-I cells produced equivalent levels of effector cytokinesas OX40 antibody costimulated OT-I cells, and would be expected to exertincreased cytotoxic activity against a target cell.

To investigate the functional activity of ImPACT+/−OX40 antibodiesversus ComPACT in a murine tumor model, CT26 cells were stablytransfected with these constructs as outlined for 3T3 cells in FIG. 7(FIGS. 10A-10C). In one set of experiments, mice were inoculated withCT26 cells on day 0, and then treated with mitomycin-C treated CT26cells, CT26-gp96-Ig, CT26-gp96-Ig combined with OX40 agonist antibodiesor with CT26 ComPACT on days 6 and 11 post tumor inoculation. In asecond set of experiments, mice were inoculated with CT26 cells on day0, and then immunized with mitomycin-C treated CT26 cells, CT26-ImPACT,CT26-ImPACT combined with OX40 agonist antibody or with CT26-ComPACTcells on days 4, 7 and 10 post tumor inoculation (FIG. 11A).Quantitative RT-PCR on tumor tissue isolated on day 12 post-tumorinoculation revealed increased expression of CD8a, IL-2 and IFNγ in OX40agonist antibody, ImPACT, ComPACT and ImPACT+OX40 agonist antibodycombination treated groups, indicating immune cell activation and tumorinfiltration. As expected, only mice receiving OX40 agonist antibodies(either alone or together with ImPACT) showed increased CD4 and FoxP3expression within the tumor (FIG. 11B). CT26 antigen-specificCD8+expansion, as detected by AH1-tetramer staining, was significantlyelevated approximately 4-fold in ImPACT+OX40 antibody and approximately5-fold in ComPACT treated mice compared with the untreated group (FIG.11B). Tumor progression was shown to be strongly blocked in micereceiving either ImPACT+OX40 agonist or ComPACT treatments as comparedto the control or monotherapy arms (FIG. 11D). This led to a significantincrease in long-term survival and a higher rate of complete tumorrejection in ComPACT treated mice (FIG. 11E, 80% and approximately 47%,respectively) compared to what we observed with the B16.F10 tumor model.Accordingly, ComPACT generates potent antigen-specific T cell expansionand tumor infiltration, delays in tumor growth and significant survivalbenefits.

The B16.F10 mouse melanoma tumor model is an aggressive tumor and is nottypically treated effectively with OX40 agonist antibody. In order toassess gp96-Ig based vaccines in the B16.F10 tumor model, a B16.F10-ovacell line was generated. In addition, B16.F10-ova-ImPACT and -ComPACTvaccines were subsequently generated by stable transfection of gp96-Igand gp96-Ig-Fc-OX40L vectors, respectively. Comparable levels of gp96-Igsecretion from the B16.F10-ImPACT and -ComPACT cell lines were confirmedby ELISA and Fc-OX40L expression in the B16.F10-ova-ComPACT cell linewas also confirmed by qRT-PCR. Mice were adoptively transferred withOT-I cells a day prior to B16.F10-ova tumor inoculation (indicating day−1, FIG. 12A). Next, the antigen specific response of OT-I cells wasinvestigated in mice following vaccination on days 4, 7 and 10 withmitomycin-C treated B16.F10-ova cells, B16.F10-ova-ImPACT,B16.F10-ova-ImPACT combined with OX40 agonist antibodies or withB16.F10-ova-ComPACT (FIG. 12B). Consistent with the data obtained withthe 3T3-ova model system, B16.F10-ova-ComPACT treated mice exhibited arobust expansion of OT-I cells between days 10 and 19 (whichcorresponded to days 6 through 15 following the initial vaccination),that was greater than that seen with ImPACT+/−OX40 agonist antibodies,with similar durable kinetics in the contraction phase to what wasobserved previously. Accordingly, B16.F10-ova-ComPACT vaccinated micedisplayed a more potent anti-tumor effect than both ImPACT+/−OX40agonist antibody vaccinations (FIG. 12C). The long term survival inComPACT treated mice was approximately 78%, with 11% of the micedemonstrating complete rejection of their aggressive tumors. Incomparison ImPACT alone vaccinated mice and ImPACT+OX40 agonist Abtreated mice showed overall survival rates of 50% and 62.5%,respectively (FIG. 12D).

The functional activities of additional ComPACTs were investigated usingthe previously described immunization assay as well as the OT-I transferassay. Specifically, OT-1/GFP cells were analyzed by flow cytometry inmice treated with No Vaccine, Ova only control cells, ComPACT(gp96-Ig/OX40L or gp96-Ig/TL1A) or ComPACT² (gp96-Ig/OX40L+TL1A), whichis a mixture of a ComPACT-OX40L cell line and a ComPACT-TL1A cell line,over 46 days, with initial vaccination on day 0 and a boost on day 35(FIG. 13). Both prime and memory responses were strong in mice treatedwith ComPACT (gp96-Ig/OX40L or gp96-Ig/TL1A) or ComPACT²(96-Ig/OX40L+TL1A). ComPACT or ComPACT² mice also surprisingly retainedelevated OT-1 levels throughout the time-course (˜days 7-20).Additionally, C57BL/6 mice were immunized with ImPACT alone or ComPACT(gp96-OX40L, gp96-Ig/4-1BBL or gp96-Ig/ICOS-L) at day 0 (FIG. 14).Results indicate that the various ComPACTS enhanced the proliferation ofOTI cells compared to ImPACT.

The in vivo activities of the additional ComPACTS were furtherinvestigated in the CT26 colorectal carcinoma model. Specifically, micewere either untreated or vaccinated on days 4, 7 and 10 with CT26parental cells, ImPACT alone, ImPACT+the TNFRSF25 agonist (4C12 ab),4C12 (ab) alone, PD-1 (ab) alone, 4C12 (ab) and PD-1 (ab), ComPACT(gp96-Ig/OX40L or gp96-Ig/TL1A), ComPACT (gp96-Ig/OX40L)+PD-1 (ab), orComPACT² (gp96-Ig/OX40L+TL1A) (FIG. 15). Results indicate that ComPACTtreatment alone (gp96-Ig/OX40L or gp96-Ig/TL1A) and in combination withPD-1 significantly reduced tumor growth. As shown in FIG. 16, ComPACTtreatment alone (gp96-Ig/OX40L or gp96-Ig/TL1A) and in combination withPD-1 also significantly enhanced mice survival.

Expression of ComPACT in human cancer cell lines was tested.Specifically, ComPACT (gp96-Ig/OX40L) was transfected into a humanprostate cancer cell line (e.g., PC-3) or a human lung adenocarcinomacell line (e.g., AD100). See FIGS. 17 and 18, respectively. Resultsindicate that both cell lines produced and excreted OX40L.

Altogether, these data demonstrated that combination immunotherapy maybe approached by incorporating multiple complementary modalities, inthis case a vaccine and T cell costimulatory fusion protein, in a singlecompound. Provision of T cell costimulation by vector-encoded andcell-secreted Fc-OX40L was feasible, and led to enhanced proliferationof antigen-specific CD8+ T cells at the time of both priming andboosting as compared to OX40 agonistic antibodies. T cells activated bythe combined vaccine and costimulator produced IFNγ, IL-2, TNFα, andgranzyme-B, and were not accompanied by off-target T cell proliferationand systemic inflammatory cytokine increases observed with OX40 agonistantibodies. Importantly, this approach also enhanced therapeutic tumorimmunity in an established murine colon cancer model. Together, theseresults provide a strategy for implementing combination immunotherapythat may not rely on double or triple antibody combinations, and whichmay provide greater safety and efficacy for patients due to reducedoff-target T cell activation.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The content of any individual section may be equally applicable to allsections.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in anyway.

The invention claimed is:
 1. An expression vector comprising a firstnucleotide sequence that encodes a secretable vaccine protein, and asecond nucleotide sequence that encodes a T cell costimulatory fusionprotein comprising 4-1BBL-Ig, wherein: the 4-1BBL-Ig fusion proteincomprises an amino acid sequence having at least 97% identity with SEQID NO: 7 and enhances activation of antigen-specific T cells whenadministered to a subject.
 2. The expression vector of claim 1, whereinthe vector is a mammalian expression vector.
 3. The expression vector ofclaim 1, wherein the secretable vaccine protein is a gp96-Ig fusionprotein that lacks the gp96 KDEL (SEQ ID NO: 3) sequence.
 4. Theexpression vector of claim 3, wherein the Ig tag in the gp96-Ig fusionprotein comprises the Fc region of human IgG1, IgG2, IgG3, or IgG4. 5.The expression vector of claim 1, wherein the expression vectorcomprises DNA.
 6. The expression vector of claim 1, wherein theexpression vector comprises RNA.
 7. The expression vector of claim 1,wherein the expression vector is incorporated into a virus or virus-likeparticle.
 8. The expression vector of claim 1, wherein the expressionvector is incorporated into a human tumor cell.
 9. The expression vectorof claim 8, wherein the human tumor cell is a cell from an establishedNSCLC, bladder cancer, melanoma, ovarian cancer, renal cell carcinoma,prostate carcinoma, sarcoma, breast carcinoma, squamous cell carcinoma,head and neck carcinoma, hepatocellular carcinoma, pancreatic carcinoma,or colon carcinoma cell line.
 10. The expression vector of claim 1,wherein the secretable vaccine protein is a gp96-Iq fusion protein, andwherein gp96 of the said gp96-Ig fusion comprises the first 799 aminoacids of SEQ ID NO: 2, such that it lacks a C-terminal KDEL (SEQ ID NO:3) sequence.
 11. The expression vector of claim 10, wherein the Ig tagof said gp96-Ig fusion protein is a CH2-CH3 domain of a human IgG. 12.The expression vector of claim 11, wherein said human IgG is human IgG1.13. A human tumor cell comprising the expression vector of claim 1.